Development and validation of the Flare-OA questionnaire for measuring flare in knee and hip osteoarthritis.

OBJECTIVE: Ability to assess flares in osteoarthritis (OA) of the knee and hip (KHOA) is important in clinical care and research. Using mixed methods, we developed a self-reported instrument measuring flare and assessed its psychometric properties. METHODS: We constructed questionnaire items from semi-structured interviews and a focus group (patients, clinicians) by using a dual-language (English-French) approach. A Delphi consensus method was used to select the most relevant items. Patients with OA from Australia, France and the United States completed the preliminary Flare-OA, HOOS, KOOS and Mini-OAKHQOL questionnaires online. We used a factor analysis and content approach to reduce items and determine structural validity. We tested the resulting questionnaire (score 0-100) for internal consistency, convergent and known-groups validity. RESULTS: Initially, 180 statements were generated and reduced to 33 items in five domains (response 0 = not at all, to 10 = absolutely) by Delphi consensus (50 patients, 116 professionals) and an expert meeting. After 398 patients (mean [SD] age 64 [8.5] years, 70.4% female, 86.7% knee OA) completed the questionnaire, it was reduced to 19 items by factor analysis and a content approach (RMSEA = 0.06; CFI = 0.96; TLI = 0.94). The Cronbach's alpha was >0.9 for the five domains and the whole questionnaire. Correlation coefficients between Flare-OA and other instrument scores were as predicted, supporting construct validity. The difference in Flare-OA score between patients with and without flare (31.8) largely exceeded 2 SEM (10.2). CONCLUSION: Flare-OA is a valid and reliable patient-reported instrument for assessing the occurrence and severity of flare in patients with KHOA in clinical research.

Outcomes for Pressure Ulcer Trials (OUTPUTs) project: review and classification of outcomes reported in pressure ulcer prevention research.

In order to overcome inconsistencies in the reporting of outcomes in clinical trials, core outcome sets (COSs) have been developed in many clinical areas and the awareness of this concept is growing steadily. The Outcomes for Pressure Ulcer Trials (OUTPUTs) project aims to improve the quality of evidence from pressure ulcer prevention trials by developing a COS. As an initial step in the COS process we aimed to identify and classify both outcomes and concepts that represent potential outcomes for future trials that have been reported in pressure ulcer prevention research. A review was conducted in 12 major databases covering the literature indexed until 2016. Outcomes and relevant concepts reported in primary studies and/or reviews on pressure ulcer prevention in adult patients were extracted as presented in the articles, and afterwards inductively grouped into outcome domains. The domains were then categorized according to the outcome domain taxonomy recently proposed by the COMET group. In total 332 studies were included and 68 outcome domains were identified, covering multiple aspects of pressure ulcer prevention. Pressure ulcer occurrence was reported in 71% of all included studies, representing the most frequent outcome, followed by costs (22% of all studies) and acceptability of intervention and comfort (18% of all studies). A plethora of different outcomes are applied in pressure ulcer prevention research and substantial variations in definitions and reporting of similar outcomes were observed. A COS for pressure ulcer prevention trials is needed to overcome the noncomparability of outcomes.

Systematic review of clinical practice guidelines for colorectal and anal cancer: the extent of recommendations for managing long-term symptoms and functional impairments.

PURPOSE: Due to increasing numbers of colorectal and anal cancer survivors, more individuals are living with long-term symptoms after treatment. A systematic review was undertaken to assess the extent to which practice guidelines for colorectal and anal cancer provide recommendations for managing long-term symptoms and functioning impairments. METHODS: Four electronic databases and websites of 30 international cancer societies were searched for clinical practice guidelines, consensus statements, or best practice recommendations for colorectal or anal cancer. Quality of included guidelines was evaluated with the Appraisal of Guidelines for Research & Evaluation II tool. Results were narratively summarized. RESULTS: We included 51 guidelines or consensus statements. Recommendations for managing long-term symptoms or functioning impairments were reported in 13 guidelines (25.4%). All 13 recommend a healthy lifestyle, diet, body weight, and physical activity. The ASCO Colorectal Cancer Survivorship Care Guideline is the most comprehensive, including interventions targeting sexual and bowel function to pain and cognitive issues, and also highlights limited evidence for informing management strategies. Other guidelines recommend treating incontinence, chronic diarrhea, and distress, and stress the need for greater awareness for sexual dysfunction, survivorship clinics, and referrals to specific supportive care interventions. CONCLUSIONS: Few clinical practice guidelines include recommendations for managing long-term symptoms and functioning impairments. It is unclear if this is due to limited evidence or absence of management strategies and interventions. Clear recommendations for managing long-term symptoms and functioning to help health professionals in supporting colorectal and anal cancer survivors are needed.

A systematic review of the impact of contemporary treatment modalities for cervical cancer on women's self-reported health-related quality of life.

PURPOSE: Given the high survival rate of cervical cancer patients, understanding women's health-related quality of life (HRQL) during and after treatment is of major clinical importance. We conducted a systematic review to synthesize all available evidence about the effects of each contemporary treatment modality for cervical cancer on all dimensions of women's HRQL, including symptoms, functioning, and global HRQL. METHODS: We searched four electronic databases from January 2000 to September 2019, cross-referenced and searched by author name for studies of patients treated for cervical cancer that reported patient-reported outcomes (PROs) before treatment and with at least one post-treatment measurement. Two independent reviewers applied inclusion and quality criteria and extracted findings. Studies were categorized by treatment to determine specific treatment effects on PROs. Results were narratively summarized. RESULTS: We found twenty-nine papers reporting 23 studies. After treatments with curative intent for early or locally advanced disease, lymphedema, diarrhea, menopausal symptoms, tight and shorter vagina, pain during intercourse, and sexual worries remained long-term problems; however, sexual activity improved over time. HRQL and psychological distress were impacted during treatment with also worsening of global HRQL but improved 3-6 months after treatment. In patients with metastatic or recurrent disease, pain improved during palliative treatment or remained stable, with no differences in global HRQL found over time. CONCLUSION: Whereas most symptoms worsen during treatment and improve in the first 3 months after completing treatment, symptoms like lymphedema, menopausal symptoms, and sexual worries develop gradually and persist after curative treatment. These findings can be used to inform clinical practice and facilitate communication and shared decision-making. More research is needed in very early cervical cancer and the impact of fertility sparing therapy on PROs.

International validation of the European Organisation for Research and Treatment of Cancer QLQ-BRECON23 quality-of-life questionnaire for women undergoing breast reconstruction.

BACKGROUND: The aim was to carry out phase 4 international field-testing of the European Organisation for Research and Treatment of Cancer (EORTC) breast reconstruction (BRECON) module. The primary objective was finalization of its scale structure. Secondary objectives were evaluation of its reliability, validity, responsiveness, acceptability and interpretability in patients with breast cancer undergoing mastectomy and reconstruction. METHODS: The EORTC module development guidelines were followed. Patients were recruited from 28 centres in seven countries. A prospective cohort completed the QLQ-BRECON15 before mastectomy and the QLQ-BRECON24 at 4-8 months after reconstruction. The cross-sectional cohort completed the QLQ-BRECON24 at 1-5 years after reconstruction, and repeated this 2-8 weeks later (test-retest reliability). All participants completed debriefing questionnaires. RESULTS: A total of 438 patients were recruited, 234 in the prospective cohort and 204 in the cross-sectional cohort. A total of 414 reconstructions were immediate, with a comparable number of implants (176) and donor-site flaps (166). Control groups comprised patients who underwent two-stage implant procedures (72, 75 per cent) or delayed reconstruction (24, 25 per cent). Psychometric scale validity was supported by moderate to high item-own scale and item-total correlations (over 0·5). Questionnaire validity was confirmed by good scale-to-sample targeting, and computable scale scores exceeding 50 per cent, except nipple cosmesis (over 40 per cent). In known-group comparisons, QLQ-BRECON24 scales and items differentiated between patient groups defined by clinical criteria, such as type and timing of reconstruction, postmastectomy radiotherapy and surgical complications, with moderate effect sizes. Prospectively, sexuality and surgical side-effects scales showed significant responsiveness over time (P < 0·001). Scale reliability was supported by high Cronbach's α coefficients (over 0·7) and test-retest (intraclass correlation more than 0·8). One item (finding a well fitting bra) was excluded based on high floor/ceiling effects, poor test-retest and weak correlations in factor analysis (below 0·3), thus generating the QLQ-BRECON23 questionnaire. CONCLUSION: The QLQ-BRECON23 is an internationally validated tool to be used alongside the EORTC QLQ-C30 (cancer) and QLQ-BR23 (breast cancer) questionnaires for evaluating quality of life and satisfaction after breast reconstruction.

Invasive lobular carcinoma of the breast; clinicopathologic profile and response to neoadjuvant chemotherapy over a 15-year period.

INTRODUCTION: Invasive lobular carcinoma (ILC) accounts for 5-15% of invasive breast cancers. Typical ILC is oestrogen receptor (ER) positive and human epidermal growth factor receptor 2 (HER2) negative. Atypical biomarker profiles (ER- and HER2+, ER+ and HER2+ or triple negative) appear to differ from typical ILCs. This study compared subtypes of ILC in terms of clinical and pathological parameters, and response to neoadjuvant chemotherapy (NACT) according to biomarker profile. METHODS: All patients with ILC treated in a single centre from January 2005 to December 2020 were identified from a prospectively maintained database. Clinicopathologic and outcome data was collected and analysed according to tumour biomarker profile. RESULTS: A total of 582 patients with ILC were treated. Typical ILC was observed in 89.2% (n = 519) and atypical in 10.8% (n = 63). Atypical ILCs were of a higher grade (35% grade 3 vs 9.6% grade 3, p < 0.001). A larger proportion of atypical ILC received NACT (31.7% vs 6.9% p < 0.001). Atypical ILCs showed a greater response to NACT (mean RCB (Residual Cancer Burden Score) 2.46 vs mean RCB 3.41, p = 0.0365), and higher pathological complete response rates (15% vs 0% p = 0.017). Despite this, overall 5-year disease-free survival (DFS) was higher in patients with typical ILC (91% vs 83%, p = 0.001). CONCLUSIONS: Atypical ILCs have distinct characteristics. They are more frequently of a higher grade and demonstrate a superior response to NACT. Despite the latter, atypical ILCs have a worse 5-year DFS which should be taken into consideration in terms of prognostication and may assist patient selection for NACT.

'PartBreCon' study. A UK multicentre retrospective cohort study to assess outcomes following PARTial BREast reCONstruction with chest wall perforator flaps.

BACKGROUND: Partial breast reconstruction with a pedicled chest wall perforator flap (CWPF) enables breast conservation in a higher tumour: breast volume ratio scenario. Since there is limited evidence, this retrospective cohort study aimed to ascertain immediate (30-days) and medium-term (follow-up duration) surgical outcomes. METHODS: STROBE-compliant protocol ascertained CWPF outcomes between March 2011-March 2021. UK centres known to perform CWPF were invited to participate if they performed at least 10 cases. Data were retrospectively collected, including patient demographics, tumour and treatment characteristics, and surgical and oncological outcomes. Statistical analysis (R™) included multivariable logistic regression and sensitivity analysis. RESULTS: Across 15 centres, 507 patients with median age (54 years, IQR; 48-62), body mass index (25.4 kg/m2, IQR; 22.5-29), tumour size (26 mm, IQR; 18-35), and specimen weight (62 g, IQR; 40-92) had following flap types: LiCAP (54.1%, n = 273), MiCAP/AiCAP (19.6%, n = 99), LiCAP + LTAP (19.8%, n = 100) and TDAP (2.2%, n = 11). 30-days complication rates were in 12%: haematoma (4.3%, n = 22), wound infection (4.3%, n = 22), delayed wound healing (2.8%, n = 14) and flap loss (0.6%, n = 3; 1 full) leading to readmissions (2.6%, n = 13) and re-operations (2.6%, n = 13). Positive margins (n = 88, 17.7%) led to 15.9% (n = 79) re-excisions, including 7.5% (n = 37) at the planned 2nd of 2-stage surgery and 1.8% (n = 9) mastectomy. At median 23 months (IQR; 11-39) follow-up, there were 1.2% (n = 6) symmetrisations; recurrences: local (1%), regional/nodal (0.6%) and distant (3.2%). CONCLUSIONS: This large multicentre cohort study demonstrates acceptable complication and margin re-excision rates. CWPF extends the range of breast conservation techniques. Further studies are required for long-term oncological outcomes.

A systematic literature review of the management, oncological outcomes and psychosocial implications of male breast cancer.

BACKGROUND: Although male breast cancer (MBC) is a rare disease, accounting for <1% of all breast cancers, it has significant oncological, survival and psychosocial implications for patients. The aim of this study is to assess the latest literature in the diagnosis, management, oncological outcomes, and psychosocial impact of MBC. METHODS: A systematic literature review was conducted using the PRISMA guidelines (Moher et al., 2009) [1] to explore the management of MBC, with particular focus on investigative imaging, surgical management, oncological outcomes, survival, genetic screening and psychosocial effects. Electronic databases were searched for randomised control trials, cohort studies and case series involving more than 10 patients. Imaging and surgical techniques, local and distant disease recurrence, survival, genetic screening and psychosocial implications in the setting of MBC were assessed. RESULTS: The search criteria identified 199 articles, of which 59 met the inclusion criteria. This included 39,529 patients, with a mean age of 64.5 years (55-71), and a mean follow-up of 66.3 months (26.2-115). Mastectomy remains the most frequently used surgical technique, with an average of 89.6%. Loco-regional and distant recurrence rate was 10.1% and 21.4% respectively. Disease-free survival (DFS) at 5 and 10 years was 66.8% and 54.5% respectively. Disease-specific survival (DSS) at 5 and 10 years was 87.1% and 67.1% respectively. Overall survival (OS) at 5 and 10 years was 72.7% and 50.7% respectively. Genetic screening was conducted in 38.6% of patients of which 4.8% and 15.8% were found to be BRCA1 and BRCA2 carriers respectively. Psychosocial studies were conducted mainly using questionnaire and interview-based methodology focusing primarily on awareness of breast cancer in men, support available and impact on gender identity. CONCLUSIONS: This review demonstrates that men present with later stage disease with subsequent impact on survival outcomes. There remains a paucity of high-level evidence and prospective studies are required. There is a need for increasing awareness amongst the public and health care professionals in order to improve outcomes and reduce stigma associated with MBC.

A systematic review of oncoplastic volume replacement breast surgery: oncological safety and cosmetic outcome.

INTRODUCTION: Oncoplastic breast conserving surgery allows higher volume excision to achieve oncological safety with minimal aesthetic compromise. The primary outcome of this study was to assess the oncological safety in the setting of volume replacement oncoplastic breast conserving surgery. The secondary objective was to assess cosmetic outcome. METHODS: A systematic literature review was conducted using the Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines to explore the oncological safety of oncoplastic breast conserving surgery, with particular focus on volume replacement. Resection margin rates, re-excision rates, conversion to mastectomy rates, local and distant disease recurrence, volume replacement techniques, cosmetic outcomes and patient-reported outcome measures were assessed. FINDINGS: The search criteria identified 155 articles, of which 40 met the inclusion criteria. These studies included 2,497 patients with a mean age of 47.8 years (range 38.4-59.6 years), a body mass index of 24.3kg/m2 (22.1-28.0kg/m2), with a mean follow-up of 37.1 months (6-125 months). A variety of volume replacement techniques were used, most commonly latissimus dorsi and chest wall perforator flaps. Whole mean pathological tumour size was 29.7mm (17-65mm) and mean specimen weight was 123.6g (46.5-220g). Mean re-excision rate was 7.2% and completion mastectomy rate was 2.3%. Locoregional and distant recurrence rate was 2.5% (0-8.1%) and 3.1% (0-14.6%), respectively. There were a variety of patient-reported outcome measures employed, with overall good to excellent outcomes. CONCLUSIONS: This review demonstrates that volume replacement oncoplastic breast conserving surgery is a safe option in terms of re-excision, completion mastectomy rates, and local and distant recurrence. Available patient-related outcome measures and cosmetic assessment tend towards better outcomes compared with wide local excision and mastectomy. However, data are significantly limited, with a paucity of high-level evidence, and it is therefore necessary to be cautious regarding the strength and interpretation of data in this review. Further prospective studies are required on this subject.

Amplification and aural rehabilitation in resource-constrained environments.

OBJECTIVES: This article attempts to highlight the challenges and possibilities for hearing healthcare through technology and aural rehabilitation in a resource-constrained setting, using South Africa as an example.Results and conclusionThe authors argue that it is possible to enhance service delivery by using free resources and maximising the limited existing resources. In order to provide a sustainable hearing healthcare service in developing countries, it is pertinent to understand the context where the services are needed, and not just adopt an approach developed for a different context. Audiologists in such settings need to employ strategies to develop context-specific tools, and adapt existing tools to serve the needs of the local population. Some examples, although not exhaustive, are provided in the article.

Evaluation of methods and costs for detecting methicillin-resistant Staphylococcus aureus isolates from clinical specimens at regional hospitals in Trinidad and Tobago.

OBJECTIVES: To evaluate and determine the most cost effective, rapid and specific method for detection of methicillin resistance in clinical isolates of S. aureus in a setting with limited personnel and resources. METHODS: Standard laboratory methods were used to identify S. aureus isolates. The conventional Methicillin Resistance Staphylococcus aureus (MRSA) detection methods used included, 1 microg oxacillin disk diffusion, oxacillin salt agar screen (CLSI), penicillin binding protein (PBP 2') latex agglutination test and E-tests oxacillin. Results of conventional tests were compared with a polymerase chain reaction (PCR) method for detecting MRSA isolates. Polymerase chain reaction detection of the mecA gene in S. aureus was used as the "gold standard" for MRSA identification. RESULTS: All methods had 100% sensitivity except for oxacillin disk diffusion and oxacillin-salt agar screening with 98% and 99%, respectively. Specificity was also 100% for all methods except for oxacillin-disk diffusion (99%). Turn around time (TAT) for detection of MRSA was calculated to be within six hours for PCR. The fastest TAT of 1.25 hours was obtained for PBP 2' latex agglutination. Total cost for labour and materials to perform each method was highest for E-test, US$13.76/isolate. The cost for PCR when compared to that of latex agglutination was not statistically significant (US$3.74 vs US5.91, p = 0.4). CONCLUSIONS: All methods presented high sensitivity and specificity, but the latex agglutination test had the advantage of giving a reliable, rapid and most cost effective result that compares well to PCR in this environment.

Leveraging the Family Influence of Women in Prostate Cancer Efforts Targeting African American Men.

BACKGROUND: Incidence rate of prostate cancer among African American (AA) men is 1.6 times that in White men. Prevention efforts in this population have typically been through faith-based organizations and barber shops, with a few including significant others. Culturally, women are known to have a strong influence in the AA family. The current study assessed prostate cancer knowledge and explored perceptions on the roles of women in prostate cancer prevention. METHODS: To assess prostate cancer knowledge, a 25-item questionnaire was administered to convenience samples of AA women (n = 297) and men (n = 199). Four focus groups were conducted to explore perceptions on the role of women in prostate cancer prevention. RESULTS: Men had a higher mean score (13.2; max of 25) than women (11.4) for knowledge of prostate cancer. For the men, higher knowledge scores were associated with having a family member diagnosed with prostate cancer and likelihood to engage healthcare providers about prostate cancer (p < 0.05). Themes from the focus groups included education/information resource, support and encouragement, instituting a "culture" of regular primary care, modeling healthcare-seeking behavior, surveillance and monitoring, motivation, and influencing diet. The major barrier to women engaging in the roles identified was limited knowledge. CONCLUSION: Including women in educational interventions may yield added benefits particularly in encouraging AA men to seek regular primary care. This affords men opportunities for dialog with healthcare providers about prostate cancer and informed decision making regarding screening.

Implementation of micromethods to resolve problems of human breast tumor heterogeneity in analysis of cyclic 3':5'-nucleotide phosphodiesterase.

Individual human infiltrating ductal carcinomas and fibroadenomas were sectioned frozen to yield an alternating sequence of stained and lyophilized material. Stained preparations were used as references permitting microdissection of regions of tumor involvement in the corresponding dried sections. Tissues quantities of 5 to 25 micrograms dry weight were incubated under mineral oil in reaction volumes of 5 microliters and analyzed for cyclic adenosine 3':5'-monophosphate phosphodiesterase (PDE). The observed affinity constants for the 27,000 x g soluble PDE from benign tumors were 4.7 and 49.9 microM, while those for malignant tumors were 6.3 and 28.5 microM. The soluble enzyme of both tumor types eluted in three peaks on DEAE-Sephacel microcolumns. Both tumor types possessed a PDE activator eluting at 350 mM NaCl, although endogenous PDE activities were unaffected by additions of either this activator or 200 microM ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid. Individual microsections of benign tumors contained total PDE levels 2-fold higher than those of malignant tumors. Homogenates prepared from pooled microsections of the same tumors possessed only one-half of the total activity. Differential losses of enzyme in various preparation schemes as well as the use of tumor samples differing in cell density were suggested to account for some of the apparently conflicting literature values for breast tumor PDE.

The Leeds Assessment of Neuropathic Symptoms and Signs Scale (LANSS) is not an adequate outcome measure of pressure ulcer-related neuropathic pain.

BACKGROUND: Few pain assessment scales have been used in pressure ulcer (PU) research and none developed or validated for people with PUs. We examined the Leeds Assessment of Neuropathic Symptoms and Signs (LANSS) scale to determine its utility as an outcome measure for people with pressure area-related pain. METHODS: Leeds Assessment of Neuropathic Symptoms and Signs data from 728 participants underwent psychometric analyses: traditional tests for data quality, scaling assumptions, reliability and validity and a Rasch analysis including tests of fit, spread and targeting of item locations, response dependency, person separation index (reliability) and differential item functioning. RESULTS: Our findings offer support for a unidimensional scale; confirmatory factor analysis indicated a non-significant chi-square test of model fit [(df = 14) 23.48, p = 0.053]. However, some misfit was identified at the overall scale and individual item levels, and internal construct validity of the LANSS as an outcome measure for neuropathic pain in people with pressure area-related pain was not supported; low to moderate item-total correlations [Chi Square (df = 28) 55.546, p = 0.002] and inter-item correlations (mean 0.117 and range from 0.063 to 0.415); and low Cronbach's alpha (0.549) and Person Separation Index (0.334). CONCLUSIONS: Requirements for reliable and valid measurement do not support the use of the LANSS as an outcome measure in people with PUs at the individual level or as a generalized measurement scale of neuropathic pain across ulcer severity groups. Expanding the number of items to aid differentiation between neuropathic pain levels and improving scale reliability is recommended. WHAT DOES THIS STUDY ADD?: The Leeds Assessment of Neuropathic Symptoms and Signs scale (LANSS) is not suitable as an outcome measure of pressure ulcer-related neuropathic pain as it did not meet requirements for reliable and valid measurement in this population.

What happened to enterovirus D68 infections in 2015?

BACKGROUND: Enterovirus-D68 (EV-D68) was observed in association with severe respiratory disease in children in North America and around the world in the fall of 2014. OBJECTIVE: To compare fall 2014 detection rates with fall 2015 detection rates of EV-D68 in nasopharyngeal swab (NPS) samples collected for routine clinical care from a large regional laboratory in south-central Ontario. METHOD: Consecutive NPS samples submitted from inpatients and outpatients in Hamilton, Niagara Region and Burlington to the Regional Virology Laboratory were tested with multiplex polymerase chain reaction (PCR) for rhinovirus/enterovirus (as a single target) and for other common respiratory viruses. All NPS samples positive for rhinovirus/enterovirus were reflexed to a lab-developed single target PCR for EV-D68 detection. RESULTS: In 2014, between August 1 and October 31, 566 of 1,497 (38%, 95%CI 35-40%) NPS samples were rhino/enterovirus positive, of which 177 (31%, 95%CI 27-35%) were confirmed as EV-D68. In 2015, between August 1 and October 31, 472 of 1,630 (29%, 95%CI 27-31%) NPS samples were rhino/enterovirus positive, of which none (0%, upper limit 97.5%CI 0.8%) were confirmed to be EV-D68. CONCLUSION: Based on testing results, there appears to be much less circulating EV-D68 in south central Ontario in 2015 than in 2014. Further studies would be helpful to determine if detection rates have also dramatically decreased in other regions in Canada and internationally.

Glycogen degradation during migration of presumptive cell types in Dictyostelium discoideum.

During the time course of differentiation in Dictyostelium discoideum, glycogen was found to accumulate from the amoebae stage to the culmination stage of development. Upon sorocarp formation (23 h), glycogen was rapidly degraded. Ultramicrotechniques, utilizing amplification of glycogen by enzymatic cycling, were used to follow glycogen metabolism in pre-stalk and prespore cells during the differentiation cycle. Both cell types accumulated glycogen at nearly the same rate. By the pseudoplasmodium stage of development glycogen had accumulated to 50% of its maximum value, and no differences were found between pre-stalk and pre-spore cells. Glycogen was degraded as pre-stalk cells migrated into the position for stalk construction. At the culmination stage of development stalk cells near the base were devoid of glycogen while pre-stalk cells near the apex of the stalk showed no loss of glycogen. The complete loss of glycogen from stalk cells occurred over a distance occupied by approximately 100 cells, and over a time period of approx. 1 h. Pre-spore cells at the culmination stage showed no loss of glycogen even though separated from stalk cells by only a thin cellulose sheath. The degradation of prespore cell glycogen did not commence until stalk construction was completed and the pre-spore mass had reached the apex of the stalk. Pre-spore cells at the culmination stage contained high levels of glycogen while only 2 h later, total degradation had occurred.

Temporal regulation of the Dictyostelium glycogen phosphorylase 2 gene.

The product of the glycogen phosphorylase-2 gene in Dictyostelium functions to provide the glucose units that are used to construct the structural components of the terminal stage of development. In this report, we link a 1233 bp upstream gp2 fragment to a luciferase reporter gene in order to study the sequences that are involved in the temporal expression of the gene. Various deletions of the promoter-luciferase fusion were then transformed into Dictyostelium cells. All deletion constructs, from -1216 to -486 nucleotides from the translational start codon, showed the same temporal pattern of expression as the authentic gp2 gene, as well as similar luciferase activities. Removal of an additional 37 nucleotides resulted in nearly 100-fold decrease in activity, yet retained the normal temporal expression of luciferase. Analysis of DNA binding proteins with the gel shift assay revealed a stage-dependent pattern of proteins that bound to the gp2 promoter. A similar pattern of temporal expression of the binding proteins was observed with either the full-length probe or with oligonucleotide probes that contained sequences that were identified as putative regulatory sites. Likewise, the full-length and oligonucleotide probes demonstrated identical binding patterns during several steps of purification of the DNA binding proteins. SDS-PAGE and Southwestern blot analysis of a DNA-affinity purified fraction, identified a 23 kDa peptide as the binding protein.