bluuebunny/crossref_metadata_2025 huggingface dataset:
Colorectal cancer screening among African American church members: A qualitative and quantitative study of patient-provider communication
Authors: Katz Mira L , James Aimee S , Pignone Michael P , Hudson Marlyn A , Jackson Ethel , Oates Veronica , Campbell Marci K
Published: January 2005

Background

A healthcare provider's recommendation to undergo screening has been shown to be one of the strongest predictors of completing a colorectal cancer (CRC) screening test. We sought to determine the relationship between the general quality of self-rated patient-provider communication and the completion of CRC screening.

Methods

A formative study using qualitative data from focus groups and quantitative data from a cross-sectional survey of church members about the quality of their communication with their healthcare provider, their CRC risk knowledge, and whether they had completed CRC screening tests. Focus group participants were a convenience sample of African American church members. Participants for the survey were recruited by telephone from membership lists of 12 African American churches located in rural counties of North Carolina to participate in the WATCH (Wellness for African Americans Through Churches) Project.

Results

Focus Groups. Six focus groups (n = 45) were conducted prior to the baseline survey. Discussions focused on CRC knowledge, and perceived barriers/motivators to CRC screening. A theme that emerged during each groups' discussion about CRC screening was the quality of the participants' communication with their health care provider. Survey. Among the 397 participants over age 50, 31% reported CRC screening within the recommended guidelines. Participants who self-rated their communication as good were more likely to have been screened (36%) within the recommended guidelines than were participants with poor communication (17%) (OR = 2.8, 95% CI 1.2, 6.4; p = 0.013). Participants who had adequate CRC knowledge completed CRC screening at a higher rate than those with inadequate knowledge (p = 0.011). The percentage of participants with CRC screening in the recommended guidelines, stratified by communication and knowledge group were: 42% for good communication/adequate knowledge; 27% for good communication/inadequate knowledge; 29% for poor communication/adequate knowledge; and 5% for poor communication/inadequate knowledge.

Conclusions

Participants who rated their patient-provider communication as good were more likely to have completed CRC screening tests than those reporting poor communication. Among participants reporting good communication, knowledge about colorectal cancer was also associated with test completion. Interventions to improve patient-provider communication may be important to increase low rates of CRC screening test completion among African Americans.
Patients' request for and emergency physicians' prescription of antimicrobial prophylaxis for anthrax during the 2001 bioterrorism-related outbreak
Authors: M'ikanatha Nkuchia M , Julian Kathleen G , Kunselman Allen R , Aber Robert C , Rankin James T , Lautenbach Ebbing
Published: January 2005

Background

Inappropriate use of antibiotics by individuals worried about biological agent exposures during bioterrorism events is an important public health concern. However, little is documented about the extent to which individuals with self-identified risk of anthrax exposure approached physicians for antimicrobial prophylaxis during the 2001 bioterrorism attacks in the United States.

Methods

We conducted a telephone survey of randomly selected members of the Pennsylvania Chapter of the American College of Emergency Physicians to assess patients' request for and emergency physicians' prescription of antimicrobial agents during the 2001 anthrax attacks.

Results

Ninety-seven physicians completed the survey. Sixty-four (66%) respondents had received requests from patients for anthrax prophylaxis; 16 (25%) of these physicians prescribed antibiotics to a total of 23 patients. Ten physicians prescribed ciprofloxacin while 8 physicians prescribed doxycycline.

Conclusion

During the 2001 bioterrorist attacks, the majority of the emergency physicians we surveyed encountered patients who requested anthrax prophylaxis. Public fears may lead to a high demand for antibiotic prophylaxis during bioterrorism events. Elucidation of the relationship between public health response to outbreaks and outcomes would yield insights to ease burden on frontline clinicians and guide strategies to control inappropriate antibiotic allocation during bioterrorist events.
The effect of short-duration sub-maximal cycling on balance in single-limb stance in patients with anterior cruciate ligament injury: a cross-sectional study
Authors: Ageberg Eva , Roberts David , Holmström Eva , Fridén Thomas
Published: January 2005

Background

It has previously been shown that an anterior cruciate ligament (ACL) injury may lead to impaired postural control, and that the ability to maintain postural control is decreased by fatigue in healthy subjects. To our knowledge, no studies have reported the effect of fatigue on postural control in subjects with ACL injury. This study was aimed at examining the effect of fatigue on balance in single-limb stance in subjects with ACL injury, and to compare the effects, and the ability to maintain balance, with that of a control group of uninjured subjects.

Methods

Thirty-six patients with unilateral, non-operated, non-acute ACL injury, and 24 uninjured subjects were examined with stabilometry before (pre-exercise) and immediately after (post-exercise) short-duration, sub-maximal cycling. In addition, the post-exercise measurements were compared, to evaluate the instantaneous ability to maintain balance and any possible recovery. The amplitude and average speed of center of pressure movements were registered in the frontal and sagittal planes. The paired t-test was used for the intra-group comparisons, and the independent t-test for the inter-group comparisons, with Bonferroni correction for multiple comparisons.

Results

No differences were found in the effects of exercise between the patients and the controls. Analysis of the post-exercise measurements revealed greater effects or a tendency towards greater effects on the injured leg than in the control group. The average speed was lower among the patients than in the control group.

Conclusions

The results of the present study showed no differences in the effects of exercise between the patients and the controls. However, the patients seemed to react differently regarding ability to maintain balance in single-limb stance directly after exercise than the control group. The lower average speed among the patients may be an expression of different neuromuscular adaptive strategies than in uninjured subjects.
Is DRE essential for the follow up of prostate cancer patients? A prospective audit of 194 patients
Authors: Ragavan Narasimhan , Sangar Vijay K , Gupta Sujoy , Herdman Jennifer , Matanhelia Shyam S , Watson Michael E , Blades Rosemary A
Published: January 2005

Background

Prostate cancer follow up forms a substantial part of the urology outpatient workload. Nurse led prostate cancer follow up clinics are becoming more common. Routine follow-up may involve performing DRE, which may require training.

Objectives

The aim of this audit was to assess the factors that influenced the change in the management of prostate cancer patients during follow up. This would allow us to pave the way towards a protocol driven follow up clinic led by nurse specialists without formal training in DRE.

Results

194 prostate cancer patients were seen over a period of two months and all the patients had DRE performed on at least one occasion. The management was changed in 47 patients. The most common factor influencing this change was PSA trend. A change in DRE findings influenced advancement of the clinic visit in 2 patients.

Conclusions

PSA is the most common factor influencing change in the management of these patients. Nurse specialists can run prostate cancer follow-up clinics in parallel to existing consultant clinics and reserve DRE only for those patients who have a PSA change or have onset of new symptoms. However larger studies are required involving all the subgroups of patients to identify the subgroups of patients who will require DRE routinely.
A randomized clinical trial comparing hydrocolloid, phenytoin and simple dressings for the treatment of pressure ulcers [ISRCTN33429693]
Authors: Hollisaz Mohammad Taghi , Khedmat Hossein , Yari Fatemeh
Published: January 2005

Background

Pressure sores are important and common complications of spinal cord injury. Many preventive and therapeutic approaches have been tried and new trials are evolving. One relatively recent method is application of a hydrocolloid dressing (HD). In this study we compared the therapeutic effects of HD on pressure ulcer healing with two other topical applications, phenytoin cream (PC) and simple dressing (SD).

Methods

Ninety-one stage I and stage II pressure ulcers of 83 paraplegic male victims of the Iran-Iraq war were randomly allocated to three treatment groups. Mean age and weight of the participants were 36.64 ± 6.04 years and 61.12 ± 5.08 kg, respectively. All the patients were managed in long term care units or in their homes for 8 weeks by a team of general practitioners and nurses, and the ulcer status was recorded as "Complete healing", "Partial healing", "Without improvement" and "Worsening".

Results

Complete healing of ulcers, regardless of location and stage, was better in the HD group than the PC [23/31(74.19%) vs 12/30(40%); difference: 34.19%, 95% CI = 10.85–57.52, (P < 0.01)] or the SD [23/31(74.19%) vs 8/30(26.66%); difference: 47.53%, 95% CI = 25.45–69.61, (P < 0.005)] groups. Complete healing of stage I ulcers in the HD group [11/13(85%)] was better than in the SD [5/11(45%); difference: 40%, 95% CI = 4.7–75.22, (P < 0.05)] or PC [2/9 (22%); difference: 63%, 95% CI = 29.69–96.3, (P < 0.005)] groups. Complete healing of stage II ulcer in the HD group [12/18 (67%)] was better than in the SD group [3/19(16%); difference: 51%, 95% CI = 23.73–78.26, (P < 0.005)], but not significantly different from the PC group [10/21 (48%); difference: 19%, 95% CI = -11.47–49.47, (P > 0.05)]. We performed a second analysis considering only one ulcer per patient (i.e. 83 ulcers in 83 patients). This "per patient" analysis showed that complete ulcer healing in the HD group was better than in the PC [20/28(71.4%) vs 11/28 (39.3%); difference: 32.1%, 95% CI = 7.4–56.7, (P < 0.01)] or SD [20/28(71.4%) vs 8/27 (29.6%); difference: 41.8%, 95% CI = 17.7–65.8, (P < 0.005)] groups.

Conclusion

We deduced that HD is the most effective method investigated for treating stage I and II pressure ulcers in young paraplegic men.
Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression
Authors: Dyson Michael R , Shadbolt S Paul , Vincent Karen J , Perera Rajika L , McCafferty John
Published: January 2005

Background

In the search for generic expression strategies for mammalian protein families several bacterial expression vectors were examined for their ability to promote high yields of soluble protein. Proteins studied included cell surface receptors (Ephrins and Eph receptors, CD44), kinases (EGFR-cytoplasmic domain, CDK2 and 4), proteases (MMP1, CASP2), signal transduction proteins (GRB2, RAF1, HRAS) and transcription factors (GATA2, Fli1, Trp53, Mdm2, JUN, FOS, MAD, MAX). Over 400 experiments were performed where expression of 30 full-length proteins and protein domains were evaluated with 6 different N-terminal and 8 C-terminal fusion partners. Expression of an additional set of 95 mammalian proteins was also performed to test the conclusions of this study.

Results

Several protein features correlated with soluble protein expression yield including molecular weight and the number of contiguous hydrophobic residues and low complexity regions. There was no relationship between successful expression and protein pI, grand average of hydropathicity (GRAVY), or sub-cellular location. Only small globular cytoplasmic proteins with an average molecular weight of 23 kDa did not require a solubility enhancing tag for high level soluble expression. Thioredoxin (Trx) and maltose binding protein (MBP) were the best N-terminal protein fusions to promote soluble expression, but MBP was most effective as a C-terminal fusion. 63 of 95 mammalian proteins expressed at soluble levels of greater than 1 mg/l as N-terminal H10-MBP fusions and those that failed possessed, on average, a higher molecular weight and greater number of contiguous hydrophobic amino acids and low complexity regions.

Conclusions

By analysis of the protein features identified here, this study will help predict which mammalian proteins and domains can be successfully expressed in E. coli as soluble product and also which are best targeted for a eukaryotic expression system. In some cases proteins may be truncated to minimise molecular weight and the numbers of contiguous hydrophobic amino acids and low complexity regions to aid soluble expression in E. coli.
Short description of an alternative simplified method for screening recombinant clones within the "AdEasy-System" by Duplex-PCR
Authors: Antolovic Dalibor , Koch Moritz , Bohlmann Inga , Kienle Peter , Büchler Markus , Weitz Jürgen
Published: January 2005

Background

Recombinant adenoviral vectors are highly efficient for in vitro and in vivo gene delivery. They can easily be produced in large numbers, transduce a wide variety of cell types and generate high levels of transgene expression.

The AdEasy system is a widely used system for generating recombinant adenoviral vectors, which are created with a minimum of enzymatic manipulations and by employing homologous recombination in E. coli.

In this paper we describe an alternative simplified method for screening recombinant DNA within the AdEasy system. This Duplex-PCR-method is independent of the transgene or insert and can be used for the complete AdEasy system. It is characterized by a simple standard protocol and the results can be obtained within a few hours. The PCR is run with two different primer sets. The primers KanaFor and KanaRev hybridizise with the Kanamycin resistence gene and AdFor and AdRev detect the adenoviral backbone. In case of recombinant clones, two diagnostic fragments with a size of 384 bp and 768 bp are generated.

Results

The practicability of this method was verified with three different transgenes: Cytosin Deaminase (AdCD), p53 (Adp53) and Granulocyte Macrophage Colony Stimulating Factor (AdGM-CSF). Recombinant clones are indicated by two diagnostic fragments and are then suitable for further processing.

Conclusion

In summary, the presented protocol allows fast detection of recombinants with an easy technique by minimizing the amount of necessary steps for generating a recombinant adenovirus. This method is time sparing and cost-effective.
Redesigned and chemically-modified hammerhead ribozymes with improved activity and serum stability
Authors: Hendry Philip , McCall Maxine J , Stewart Tom S , Lockett Trevor J
Published: January 2005

Background

Hammerhead ribozymes are RNA-based molecules which bind and cleave other RNAs specifically. As such they have potential as laboratory reagents, diagnostics and therapeutics. Despite having been extensively studied for 15 years or so, their wide application is hampered by their instability in biological media, and by the poor translation of cleavage studies on short substrates to long RNA molecules. This work describes a systematic study aimed at addressing these two issues.

Results

A series of hammerhead ribozyme derivatives, varying in their hybridising arm length and size of helix II, were tested in vitro for cleavage of RNA derived from the carbamoyl phosphate synthetase II gene of Plasmodium falciparum. Against a 550-nt transcript the most efficient (t1/2 = 26 seconds) was a miniribozyme with helix II reduced to a single G-C base pair and with twelve nucleotides in each hybridising arm. Miniribozymes of this general design were targeted to three further sites, and they demonstrated exceptional cleavage activity. A series of chemically modified derivatives was prepared and examined for cleavage activity and stability in human serum. One derivative showed a 103-fold increase in serum stability and a doubling in cleavage efficiency compared to the unmodified miniribozyme. A second was almost 104-fold more stable and only 7-fold less active than the unmodified parent.

Conclusion

Hammerhead ribozyme derivatives in which helix II is reduced to a single G-C base pair cleave long RNA substrates very efficiently in vitro. Using commonly available phosphoramidites and reagents, two patterns of nucleotide substitution in this derivative were identified which conferred both good cleavage activity against long RNA targets and good stability in human serum.
Prevalence and determinants of diabetes mellitus among Iranian patients with chronic liver disease
Authors: Alavian Seyed M , Hajarizadeh Behzad , Nematizadeh Fariborz , Larijani Bagher
Published: January 2005

Background

Alterations in carbohydrate metabolism are frequently observed in cirrhosis. We conducted this study to define the prevalence of diabetes mellitus (DM) and impaired glucose tolerance (IGT) in Iranian patients with chronic liver disease (CLD), and explore the factors associated with DM in these patients.

Methods

One hundred and eighty-five patients with CLD were enrolled into the study. Fasting plasma glucose and two-hour plasma glucose were measured in patients' sera. DM and IGT were diagnosed according to the latest American Diabetes Association criteria.

Results

The subjects included 42 inactive HBV carriers with a mean age of 42.2 ± 12.0 years, 102 patients with HBV or HCV chronic hepatitis with a mean age of 41.2 ± 10.9 years, and 41 cirrhotic patients with a mean age of 52.1 ± 11.4 years. DM and IGT were diagnosed in 40 (21.6%) and 21 (11.4%) patients, respectively. Univariate analysis showed that age (P = 0.000), CLD status (P = 0.000), history of hypertension (P = 0.007), family history of DM (P = 0.000), and body mass index (BMI) (P = 0.009) were associated with DM. Using Multivariate analysis, age (OR = 4.7, 95%CI: 1.8–12.2), family history of DM (OR = 6.6, 95%CI: 2.6–17.6), chronic hepatitis (OR = 11.6, 95%CI: 2.9–45.4), and cirrhosis (OR = 6.5, 95%CI: 2.4–17.4) remained as the factors independently associated with DM. When patients with cirrhosis and chronic hepatitis were analyzed separately, higher Child-Pugh's score in cirrhotic patients (OR = 9.6, 95%CI: 1.0–88.4) and older age (OR = 7.2, 95%CI: 1.0–49.1), higher fibrosis score (OR = 59.5, 95%CI: 2.9–1211.3/ OR = 11.9, 95%CI: 1.0–132.2), and higher BMI (OR = 30.3, 95%CI: 3.0–306.7) in patients with chronic hepatitis were found to be associated with higher prevalence of DM.

Conclusions

Our findings indicate that patients with cirrhosis and chronic hepatitis are at the increased risk of DM occurrence. Older age, severe liver disease, and obesity were associated with DM in these patients.
Tissue eosinophilia: a morphologic marker for assessing stromal invasion in laryngeal squamous neoplasms
Authors: Said Mahmoud , Wiseman Sam , Yang Jun , Alrawi Sadir , Douglas Wade , Cheney Richard , Hicks Wesley , Rigual Nestor , Loree Thom , Spiegel Gregory , Tan Dongfeng
Published: January 2005

Background

The assessment of tumor invasion of underlying benign stroma in neoplastic squamous proliferation of the larynx may pose a diagnostic challenge, particularly in small biopsy specimens that are frequently tangentially sectioned. We studied whether thresholds of an eosinophilic response to laryngeal squamous neoplasms provides an adjunctive histologic criterion for determining the presence of invasion.

Methods

Eighty-seven(n = 87) cases of invasive squamous cell carcinoma and preinvasive squamous neoplasia were evaluated. In each case, the number of eosinophils per high power field(eosinophils/hpf), and per 10 hpf in the tissue adjacent to the neoplastic epithelium, were counted and tabulated. For statistical purposes, the elevated eosinophils were defined and categorized as: focally and moderately elevated (5–9 eos/hpf), focally and markedly increased(>10/hpf), diffusely and moderately elevated(5–19 eos/10hpf), and diffusely and markedly increased (>20/10hpf).

Results

In the invasive carcinoma, eosinophil counts were elevated focally and /or diffusely, more frequently seen than in non-invasive neoplastic lesions. The increased eosinophil counts, specifically >10hpf, and >20/10hpf, were all statistically significantly associated with stromal invasion. Greater than 10 eosinophils/hpf and/or >20 eosinophils/10hpf had highest predictive power, with a sensitivity, specificity and positive predictive value of 82%, 93%, 96% and 80%, 100% and 100%, respectively. Virtually, greater than 20 eosinophils/10 hpf was diagnostic for tumor invasion in our series.

Conclusion

Our study suggests for the first time that the elevated eosinophil count in squamous neoplasia of the larynx is a morphologic feature associated with tumor invasion. When the number of infiltrating eosinophils exceeds 10/hpf and or >20/10 hpf in a laryngeal biopsy with squamous neoplasia, it represents an indicator for the possibility of tumor invasion. Similarly, the presence of eosinophils meeting these thresholds in an excisional specimen should prompt a thorough evaluation for invasiveness, when evidence of invasion is absent, or when invasion is suspected by conventional criteria in the initial sections.
Evaluation of a communication skills seminar for students in a Japanese medical school: a non-randomized controlled study
Authors: Mukohara Kei , Kitamura Kazuya , Wakabayashi Hideki , Abe Keiko , Sato Juichi , Ban Nobutaro
Published: January 2005

Background

Little data exist for the effectiveness of communication skills teaching for medical students in non-English speaking countries. We conducted a non-randomized controlled study to examine if a short intensive seminar for Japanese medical students had any impact on communication skills with patients.

Methods

Throughout the academic year 2001–2002, a total of 105 fifth-year students (18 groups of 5 to 7 students) participated, one group at a time, in a two-day, small group seminar on medical interviewing. Half way through the year, a five-station objective structured clinical examination (OSCE) was conducted for all fifth-year students. We videotaped all the students' interaction with a standardized patient in one OSCE station that was focused on communication skills. Two independent observers rated the videotapes of 50 students who had attended the seminar and 47 who had not. Sixteen core communication skills were measured. Disagreements between raters were resolved by a third observer's rating.

Results

There was a statistically significant difference in proportions of students who were judged as 'acceptable' in one particular skill related to understanding patient's perspectives: asking how the illness or problems affected the patient's life, (53% in the experimental group and 30% in the control group, p = .02). No differences were observed in the other 15 core communication skills, although there was a trend for improvement in the skill for asking the patient's ideas about the illness or problems (60% vs. 40%, p = .054) and one of the relationship building skills; being attentive and empathic nonverbally (87% vs. 72%, p = .064).

Conclusion

The results of this study suggest that a short, intensive small group seminar for Japanese medical students may have had a short-term impact on specific communication skills, pertaining to understanding patient's perspectives.
Design and validation of the Health Professionals' Attitudes Toward the Homeless Inventory (HPATHI)
Authors: Buck David S , Monteiro F Marconi , Kneuper Suzanne , Rochon Donna , Clark Dana L , Melillo Allegra , Volk Robert J
Published: January 2005

Background

Recent literature has called for humanistic care of patients and for medical schools to begin incorporating humanism into medical education. To assess the attitudes of health-care professionals toward homeless patients and to demonstrate how those attitudes might impact optimal care, we developed and validated a new survey instrument, the Health Professional Attitudes Toward the Homeless Inventory (HPATHI). An instrument that measures providers' attitudes toward the homeless could offer meaningful information for the design and implementation of educational activities that foster more compassionate homeless health care. Our intention was to describe the process of designing and validating the new instrument and to discuss the usefulness of the instrument for assessing the impact of educational experiences that involve working directly with the homeless on the attitudes, interest, and confidence of medical students and other health-care professionals.

Methods

The study consisted of three phases: identifying items for the instrument; pilot testing the initial instrument with a group of 72 third-year medical students; and modifying and administering the instrument in its revised form to 160 health-care professionals and third-year medical students. The instrument was analyzed for reliability and validity throughout the process.

Results

A 19-item version of the HPATHI had good internal consistency with a Cronbach's alpha of 0.88 and a test-retest reliability coefficient of 0.69. The HPATHI showed good concurrent validity, and respondents with more than one year of experience with homeless patients scored significantly higher than did those with less experience. Factor analysis yielded three subscales: Personal Advocacy, Social Advocacy, and Cynicism.

Conclusions

The HPATHI demonstrated strong reliability for the total scale and satisfactory test-retest reliability. Extreme group comparisons suggested that experience with the homeless rather than medical training itself could affect health-care professionals' attitudes toward the homeless. This could have implications for the evaluation of medical school curricula.
Variations and voids: the regulation of human cloning around the world
Authors: Pattinson Shaun D , Caulfield Timothy
Published: January 2005

Background

No two countries have adopted identical regulatory measures on cloning. Understanding the complexity of these regulatory variations is essential. It highlights the challenges associated with the regulation of a controversial and rapidly evolving area of science and sheds light on a regulatory framework that can accommodate this reality.

Methods

Using the most reliable information available, we have performed a survey of the regulatory position of thirty countries around the world regarding the creation and use of cloned embryos (see Table 1). We have relied on original and translated legislation, as well as published sources and personal communications. We have examined the regulation of both reproductive cloning (RC) and non-reproductive cloning (NRC).

Results

While most of the countries studied have enacted national legislation, the absence of legislation in seven of these countries should not be equated with the absence of regulation. Senator Morin was not correct in stating that the majority of recent legislation bans both RC and NRC. Recent regulatory moves are united only with regard to the banning of RC. While NRC is not permitted in seventeen of the countries examined, it could be permitted in up to thirteen countries.

Conclusions

There is little consensus on the various approaches to cloning laws and policies, and the regulatory position in many countries remains uncertain.
The effectiveness of metal on metal hip resurfacing: a systematic review of the available evidence published before 2002
Authors: Wyness Laura , Vale Luke , McCormack Kirsty , Grant Adrian , Brazzelli Miriam
Published: January 2005

Background

Conventional total hip replacement (THR) may be felt to carry too high a risk of failure over a patient's lifetime, especially in young people. There is increasing interest in metal on metal hip resurfacing arthroplasty (MoM) as this offers a bone-conserving option for treating those patients who are not considered eligible for THR. We aim to evaluate the effectiveness of MoM for treatment of hip disease, and compare it with alternative treatments for hip disease offered within the UK.

Methods

A systematic review was carried out to identify the relevant literature on MoM published before 2002. As watchful waiting and total hip replacement are alternative methods commonly used to alleviate the symptoms of degenerative joint disease of the hip, we compared MoM with these.

Results

The data on the effectiveness of MoM are scarce, as it is a relatively new technique and at present only short-term results are available.

Conclusion

It is not possible to make any firm conclusions about the effectiveness of MoM based on these early results. While the short-term results are promising, it is unclear if such results would be replicated in more rigorous studies, and what the long-term performance might be. Further research is needed which ideally should involve long-term randomised comparisons of MoM with alternative approaches to the clinical management of hip disease.
Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli
Authors: Sørensen Hans Peter , Mortensen Kim Kusk
Published: January 2005

Pure, soluble and functional proteins are of high demand in modern biotechnology. Natural protein sources rarely meet the requirements for quantity, ease of isolation or price and hence recombinant technology is often the method of choice. Recombinant cell factories are constantly employed for the production of protein preparations bound for downstream purification and processing. Eschericia coli is a frequently used host, since it facilitates protein expression by its relative simplicity, its inexpensive and fast high density cultivation, the well known genetics and the large number of compatible molecular tools available. In spite of all these qualities, expression of recombinant proteins with E. coli as the host often results in insoluble and/or nonfunctional proteins. Here we review new approaches to overcome these obstacles by strategies that focus on either controlled expression of target protein in an unmodified form or by applying modifications using expressivity and solubility tags.
Homologs of the Brugia malayi diagnostic antigen BmR1 are present in other filarial parasites but induce different humoral immune responses
Authors: Noordin Rahmah , Aziz Ros Azeana Abdul , Ravindran Balachandran
Published: January 2005

Background

The recombinant antigen BmR1 has been extensively employed in both ELISA and immunochromatographic rapid dipstick (Brugia Rapid) formats for the specific and sensitive detection of IgG4 antibodies against the lymphatic filarial parasites Brugia malayi and Brugia timori. In sera of individuals infected with Wuchereria bancrofti the IgG4 reactivity to BmR1 is variable, and cross-reactivity of sera from individuals infected with Onchocerca volvulus or Loa loa was observed only in single cases. In order to characterize the homologs of the BmR1 antigen in W. bancrofti (Wb-BmR1), O. volvulus (Ov-BmR1) and L. loa (Ll-BmR1) the cDNA sequences were identified, the protein expressed and the antibody reactivity of patients' sera was studied.

Methods

PCR methodology was used to identify the cDNA sequences from cDNA libraries and/or genomic DNA of W. bancrofti, O. volvulus and L. loa. The clones obtained were sequenced and compared to the cDNA sequence of BmR1. Ov-BmR1 and Ll-BmR1 were expressed in E. coli and tested using an IgG4-ELISA with 262 serum samples from individuals with or without B. malayi, W. bancrofti, O. volvulus and L. loa infections or various other parasitic infections. BmR1, Ov-BmR1 and Ll-BmR1 were also tested for reactivity with the other three IgG subclasses in patients' sera.

Results

Wb-BmR1 was found to be identical to BmR1. Ov-BmR1 and Ll-BmR1 were found to be identical to each other and share 99.7% homology with BmR1. The pattern of IgG4 recognition of all serum samples to BmR1, Ov-BmR1 and Ll-BmR1 were identical. This included weak IgG4 reactivities demonstrated by L. loa- and O. volvulus-infected patients tested with Ov-BmR1 and Ll-BmR1 (or BmR1). With respect to reactivity to other IgG subclasses, sera from O. volvulus- and L. loa-infected patients showed positive reactions (when tested with BmR1, Ov-BmR1 or Ll-BmR1 antigens) only with IgG1. No reactivity was observed with IgG2 or with IgG3. Similarly, ELISAs to detect reactivity to other anti-filarial IgG subclasses antibodies showed that sera from individuals infected with B. malayi or W. bancrofti (active infections as well as patients with chronic disease) were positive with BmR1 only for IgG1 and were negative when tested with IgG2 and with IgG3 subclasses.

Conclusions

This study demonstrates that homologs of the BmR1 antigen are present in W. bancrofti, O. volvulus and L. loa and that these antigens are highly conserved. Recognition of this antigen by patients' sera is similar with regard to IgG1, IgG2 and IgG3, but different for IgG4 antibodies. We conclude that the BmR1 antigen is suitable for detection of IgG4 antibodies in brugian filariasis. However, its homologs are not suitable for IgG4-based diagnosis of other filarial infections.
In vitro activity of antiamoebic drugs against clinical isolates of Entamoeba histolytica and Entamoeba dispar
Authors: Bansal Devendra , Sehgal Rakesh , Chawla Yogesh , Mahajan Ramesh Chander , Malla Nancy
Published: January 2005

Background

Amoebiasis is a major public health problem in tropical and subtropical countries. Although a number of antiamoebic agents are used for its treatment, yet the susceptibility data on clinical isolates of Entamoeba histolytica and Entamoeba dispar are not available. Therefore, the present study was aimed to assess the in vitro susceptibility of clinical isolates of E. histolytica and E. dispar to metronidazole, chloroquine, emetine and tinidazole.

Methods

A total of 45 clinical isolates (15 E. histolytica and 30 E. dispar) were maintained in polyxenic cultures followed by monoxenic cultures. In vitro drug sensitivity (IC50) of clinical isolates and standard reference strain of E. histolytica (HM1: IMSS) was assessed by nitro blue tetrazolium (NBT) reduction assay after exposure to various concentrations of each drug.

Results

The results showed that all clinical isolates had a higher IC50 compared to reference strain to all the four drugs. E. histolytica isolates appeared to be more susceptible [IC50 (μm) 13.2,26.3,31.2 and 12.4] compared to E. dispar isolates [IC50(μm) 15.6,28.9,32.8 and 13.2] and the reference strain of E. histolytica [IC50 (μm) 9.5, 15.5, 29.9 and 10.2] to the metronidazole, chloroquine, emetine and tinidazole respectively.

Conclusions

The results indicate that till date, Entamoeba isolates in India do not seem to be resistant to the commonly used antiamoebic drugs.
Helicobacter pylori and gastroduodenal pathology: New threats of the old friend
Authors: Ahmed Niyaz , Sechi Leonardo A
Published: January 2005

The human gastric pathogen Helicobacter pylori causes chronic gastritis, peptic ulcer disease, gastric carcinoma, and mucosa-associated lymphoid tissue (MALT) lymphoma. It infects over 50% of the worlds' population, however, only a small subset of infected people experience H. pylori-associated illnesses. Associations with disease-specific factors remain enigmatic years after the genome sequences were deciphered. Infection with strains of Helicobacter pylori that carry the cytotoxin-associated antigen A (cagA) gene is associated with gastric carcinoma. Recent studies revealed mechanisms through which the cagA protein triggers oncopathogenic activities. Other candidate genes such as some members of the so-called plasticity region cluster are also implicated to be associated with carcinoma of stomach. Study of the evolution of polymorphisms and sequence variation in H. pylori populations on a global basis has provided a window into the history of human population migration and co-evolution of this pathogen with its host. Possible symbiotic relationships were debated since the discovery of this pathogen. The debate has been further intensified as some studies have posed the possibility that H. pylori infection may be beneficial in some humans. This assumption is based on increased incidence of gastro-oesophageal reflux disease (GERD), Barrett's oesophagus and adenocarcinoma of the oesophagus following H. pylori eradication in some countries. The contribution of comparative genomics to our understanding of the genome organisation and diversity of H. pylori and its pathophysiological importance to human healthcare is exemplified in this review.
In vitro activity of vancomycin, quinupristin/dalfopristin, and linezolid against intact and disrupted biofilms of staphylococci
Authors: El-Azizi Mohamed , Rao Suma , Kanchanapoom Termkiat , Khardori Nancy
Published: January 2005

Shed cells or disrupted parts of the biofilm may enter the circulation causing serious and very hard to treat biofilm-associated infections. The activity of antimicrobial agents against the shed cells/disrupted biofilms is largely unknown.

Methods

We studied the in vitro susceptibility of intact and disrupted biofilms of thirty clinical isolates of methicillin-resistant and methicillin–susceptible Staphylococcus aureus (MRSA and MSSA) and Staphylococcus epidermidis to vancomycin, quinupristin/dalfopristin, and linezolid and compared it to that of the suspended (planktonic) cells.

Results

Bacteria in the disrupted biofilms were as resistant as those in the intact biofilms at the minimum inhibitory concentrations of the antibiotics. At higher concentrations, bacteria in the disrupted biofilms were significantly (P < 0.001) less resistant than those in the intact biofilms but more resistant than the planktonic cells. Quinupristin/dalfopristin showed the best activity against cells of the disrupted biofilms at concentrations above MICs and vancomycin, at 500 and 1,000 μg/ml, was significantly more active against the biofilms of MRSA and S. epidermidis

Conclusion

The difficulty of treating biofilm-associated infections may be attributed not only to the difficulty of eradicating the biofilm focus but also to the lack of susceptibility of cells disrupted from the biofilm to antimicrobial agents.
Prolonged treatment of genetically obese mice with conjugated linoleic acid improves glucose tolerance and lowers plasma insulin concentration: possible involvement of PPAR activation
Authors: Wargent Ed , Sennitt Matthew V , Stocker Claire , Mayes Andrew E , Brown Louise , O'Dowd Jacqueline , Wang Steven , Einerhand Alexandra WC , Mohede Inge , Arch Jonathan RS , Cawthorne Michael A
Published: January 2005

Background

Studies in rodents and some studies in humans have shown that conjugated linoleic acid (CLA), especially its trans-10, cis-12 isomer, reduces body fat content. However, some but not all studies in mice and humans (though none in rats) have found that CLA promotes insulin resistance. The molecular mechanisms responsible for these effects are unclear, and there are conflicting reports on the effects of CLA on peroxisomal proliferator-activated receptor-γ (PPARγ) activation and expression. We have conducted three experiments with CLA in obese mice over three weeks, and one over eleven weeks. We have also investigated the effects of CLA isomers in PPARγ and PPARα reporter gene assays.

Results

Inclusion of CLA or CLA enriched with its trans-10, cis-12 isomer in the diet of female genetically obese (lep
ob
/lep
ob
) mice for up to eleven weeks reduced body weight gain and white fat pad weight. After two weeks, in contrast to beneficial effects obtained with the PPARγ agonist rosiglitazone, CLA or CLA enriched with its trans-10, cis-12 isomer raised fasting blood glucose and plasma insulin concentrations, and exacerbated glucose tolerance. After 10 weeks, however, CLA had beneficial effects on glucose and insulin concentrations. At this time, CLA had no effect on the plasma TNFα concentration, but it markedly reduced the plasma adiponectin concentration. CLA and CLA enriched with either isomer raised the plasma triglyceride concentration during the first three weeks, but not subsequently. CLA enriched with its trans-10, cis-12 isomer, but not with its cis-9, trans-11 isomer, stimulated PPARγ-mediated reporter gene activity; both isomers stimulated PPARα-mediated reporter gene activity.

Conclusions

CLA initially decreased but subsequently increased insulin sensitivity in lep
ob
/lep
ob
mice. Activation of both PPARγ and PPARα may contribute to the improvement in insulin sensitivity. In the short term, however, another mechanism, activated primarily by trans-10, cis-12-CLA, which probably leads to reduced adipocyte number and consequently reduced plasma adiponectin concentration, may decrease insulin sensitivity.
Mitral valve prolapse associated with celiac artery stenosis: a new ultrasonographic syndrome?
Authors: Arcari Luciano
Published: January 2005

Background

Celiac artery stenosis (CAS) may be caused by atherosclerotic degeneration or compression exerted by the arched ligament of the diaphragm. Mitral valve prolapse (MVP) is the most common valvular disorder. There are no reports on an association between CAS and MVP.

Methods

1560 (41%) out of 3780 consecutive patients undergoing echocardiographic assessment of MVP, had Doppler sonography of the celiac tract to detect CAS.

Results

CAS was found in 57 (3.7%) subjects (23 males and 34 females) none of whom complained of symptoms related to visceral ischemia. MVP was observed in 47 (82.4%) subjects with and 118 (7.9%) without CAS (p < 0.001). The agreement between MVP and CAS was 39% (95% CI 32–49%). PSV (Peak Systolic Velocity) was the only predictor of CAS in MPV patients (OR 0.24, 95% CI 0.08–0.69) as selected in a multivariate logistic model.

Conclusion

CAS and MVP seem to be significantly associated in patients undergoing consecutive ultrasonographic screening.
Echocardiographic AV-interval optimization in patients with reduced left ventricular function
Authors: Melzer C , Borges AC , Knebel F , Richter WS , Combs W , Baumann G , Theres H
Published: January 2005

Background

Ritter's method is a tool used to optimize AV delay in DDD pacemaker patients with normal left ventricular function only. The goal of our study was to evaluate Ritter's method in AV delay-interval optimization in patients with reduced left ventricular function.

Methods

Patients with implanted DDD pacemakers and AVB III° were assigned to one of two groups according to ejection fraction (EF): Group 1 (EF > 35%) and Group 2 (EF < 35%). AV delay optimization was performed by means of radionuclide ventriculography (RNV) and application of Ritter's method.

Results

For each of the patients examined, we succeeded in defining an optimal AV interval by means of both RNV and Ritter's method. The optimal AV delay determined by RNV correlated well with the delay found by Ritter's method, especially among those patients with reduced EF. The intra-class correlation coefficient was 0.8965 in Group 1 and 0.9228 in Group 2. The optimal AV interval in Group 1 was 190 ± 28.5 ms, and 180 ± 35 ms in Group 2.

Conclusion

Ritter's method is also effective for optimization of AV intervals among patients with reduced left ventricular function (EF < 35%). The results obtained by RNV correlate well with those from Ritter's method. Individual programming of the AV interval is fundamentally essential in all cases.
Despite WT1 binding sites in the promoter region of human and mouse nucleoporin glycoprotein 210, WT1 does not influence expression of GP210
Authors: Olsson Magnus , English Milton A , Mason Jacqueline , Licht Jonathan D , Ekblom Peter
Published: January 2005

Background

Glycoprotein 210 (GP210) is a transmembrane component of the nuclear pore complex of metazoans, with a short carboxyterminus protruding towards the cytoplasm. Its function is unknown, but it is considered to be a major structural component of metazoan nuclear pores. Yet, our previous findings showed pronounced differences in expression levels in embryonic mouse tissues and cell lines. In order to identify factors regulating GP210, the genomic organization of human GP210 was analyzed in silico.

Results

The human gene was mapped to chromosome 3 and consists of 40 exons spread over 102 kb. The deduced 1887 amino acid showed a high degree of alignment homology to previously reported orthologues. Experimentally we defined two transcription initiation sites, 18 and 29 bp upstream of the ATG start codon. The promoter region is characterized by a CpG island and several consensus binding motifs for gene regulatory transcription factors, including clustered sites associated with Sp1 and the Wilms' tumor suppressor gene zinc finger protein (WT1). In addition, distal to the translation start we found a (GT)n repetitive sequence, an element known for its ability to bind WT1. Homologies for these motifs could be identified in the corresponding mouse genomic region. However, experimental tetracycline dependent induction of WT1 in SAOS osteosarcoma cells did not influence GP210 transcription.

Conclusion

Although mouse GP210 was identified as an early response gene during induced metanephric kidney development, and WT1 binding sites were identified in the promoter region of the human GP210 gene, experimental modulation of WT1 expression did not influence expression of GP210. Therefore, WT1 is probably not regulating GP210 expression. Instead, we suggest that the identified Sp binding sites are involved.