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Since its outbreak in early 2020, the COVID-19 pandemic has diverted resources from non-urgent and elective procedures, leading to diagnosis and treatment delays, with an increased number of neoplasms at advanced stages worldwide. The aims of this study were to quantify the reduction in surgical activity for indeterminate thyroid nodules during the COVID-19 pandemic; and to evaluate whether delays in surgery led to an increased occurrence of aggressive tumours. In this retrospective, international, cross-sectional study, centres were invited to participate in June 22, 2022; each centre joining the study was asked to provide data from medical records on all surgical thyroidectomies consecutively performed from Jan 1, 2019, to Dec 31, 2021. Patients with indeterminate thyroid nodules were divided into three groups according to when they underwent surgery: from Jan 1, 2019, to Feb 29, 2020 (global prepandemic phase), from March 1, 2020, to May 31, 2021 (pandemic escalation phase), and from June 1 to Dec 31, 2021 (pandemic decrease phase). The main outcomes were, for each phase, the number of surgeries for indeterminate thyroid nodules, and in patients with a postoperative diagnosis of thyroid cancers, the occurrence of tumours larger than 10 mm, extrathyroidal extension, lymph node metastases, vascular invasion, distant metastases, and tumours at high risk of structural disease recurrence. Univariate analysis was used to compare the probability of aggressive thyroid features between the first and third study phases. The study was registered on ClinicalTrials.gov, NCT05178186. Data from 157 centres (n=49 countries) on 87 467 patients who underwent surgery for benign and malignant thyroid disease were collected, of whom 22 974 patients (18 052 [78·6%] female patients and 4922 [21·4%] male patients) received surgery for indeterminate thyroid nodules. We observed a significant reduction in surgery for indeterminate thyroid nodules during the pandemic escalation phase (median monthly surgeries per centre, 1·4 [IQR 0·6-3·4]) compared with the prepandemic phase (2·0 [0·9-3·7]; p

Bifidobacteria have long been recognized as bacteria with probiotic and therapeutic features. The aim of this work is to characterize the

O objetivo com o presente trabalho foi avaliar o desempenho e a qualidade dos ovos de galinhas poedeiras Negras Avifran alimentadas com a inclusão de 0,8% de extratos vegetais pigmentantes (extrato de páprica (Capsicum annuum), de flor de marigold (Tagetes erectus) e 0,045 % de pigmentante sintético (cantaxantina) na ração. Utilizou-se 120 poedeiras Negras Avifran distribuídas em 4 tratamentos, 6 repetições com 5 aves cada, totalizando 24 parcelas experimentais. Foram realizadas análises de desempenho zootécnico, qualidade dos ovos e cor da gema do ovo através de dois métodos de cores, leque colorimétrico e calorímetro digital. A inclusão de 0,8% de pigmentantes naturais na dieta de poedeiras Negras Avifran criadas em clima quente, pode ser utilizada como forma de substituir a cantaxantina, pois proporciona boa coloração da gema do ovo, favorecendo sua comercialização, e não prejudica o desempenho e a qualidade interna e externa dos ovos (P>0,05).

We report the implementation of our in silico/synthesis pipeline by targeting the glutathione-dependent enzyme mPGES-1, a valuable macromolecular target in both cancer therapy and inflammation therapy. Specifically, by using a virtual fragment screening approach of aromatic bromides, straightforwardly modifiable by the Suzuki-Miyaura reaction, we identified 3-phenylpropanoic acid and 2-(thiophen-2-yl)acetic acid to be suitable chemical platforms to develop tighter mPGES-1 inhibitors. Among these, compounds 1c and 2c showed selective inhibitory activity against mPGES-1 in the low micromolar range in accordance with molecular modeling calculations. Moreover, 1c and 2c exhibited interesting IC50 values on A549 cell lines compared to CAY10526, selected as reference compound. The most promising compound 2c induced the cycle arrest in the G0/G1 phase at 24 h of exposure, whereas at 48 and 72 h, it caused an increase of subG0/G1 fraction, suggesting an apoptosis/necrosis effect.

Author of the Study: The COVID-19 outbreak has brought challenges to the global healthcare community. The management of upper urinary tract stones has been affected even further, with potential severe sequelae for patient’s health. Materials and Methods: We report a multicentric retrospective study involving 9 Centers regularly delivering treatment for upper tract urinary stones across the country. All Centers suffered significant limitations during the pandemic period due to government limitations. We compared the 12 months-period prior to COVID-19 (from march 1st 2019 to February 28th 2020, named as period A) with postCOVID-19 period (from march 1st, 2020 to February 28th, 2021 named as period B). Aim of the study was to compare endourological procedures for upper urinary stones during period A and the period B. This study investigated all types of surgeries delivered in both elective and emergency setting. Results: A total of 4018 procedures were collected, including 2176 procedures in period A. In period B, 1842 procedures were carried out, corresponding to a loss of 15.35% of cases (p < 0.001). Looking into elective cases, 1622 procedures were delivered in period A, compared to 1280 in period B, resulting in a loss of 342 cases corresponding to 21.81% (p = 0.001). All types of stone treatments resulted affected, including ESWL (from 487 cases in period A to 344 in period B, − 29.37%, p = 0.001), PCNLs (from 170 to 125 cases, corresponding to − 26.47%, p:0.008), retrograde surgery for renal stones (from 433 to 387 cases, − 10.63%, p = 0.008) and for ureteric stones (from 614 cases to 484, − 21.18%, p.008). Additionally, waiting lists resulted affected, with significant delays in period B. In particular, for ureteric stones, mean waiting time in period A was 61.5 days compared to 87.5 days in period B (p = 0.008). Regarding renal stones, waiting list increased from 64.74 days in period A to 85.66 days in period B for RIRS. The waiting list for percutaneous surgeries increased from 79 days to 103 days (p = 0.001). We did not find any patient which acquired COVID-19 during hospitalization for elective or urgent surgery. We also found a longer waiting list for pre-stented patients, resulting to be 86.5 days in period B compared to 64 days in period A (p < 0.005). Conclusions: Our study showed how COVID-19 caused a significant disruption in endourological services across the country. Our data underlined how less patients received treatment in a longer time. This can potentially lead to an increased risk of stone- related complications, including sepsis and kidney loss.

Background Well-being is the result of many factors such as health, happiness, expectations, values. Italy has been one of European countries that in recent years has suffered most combination of economic, political and social stress, with consequences on well-being. The aims of study are i) to examine health and happiness perceptions of young adults ii) to compare actual results with a previous survey conducted in 2004. Methods A descriptive cross-sectional study was conducted between Jan-Dec 2017. A validated questionnaire was administered from the Italian Centre for Social Investment (CENSIS) to 1,003 young Italians, aged 18-40. The anonymous questionnaire investigated emotional aspects, health status, social life and socioeconomic status. The statistical analysis (chi-square p Results The sample resulted quite happy (2017 vs 2004:56.1% vs 63.7%), the happiness increasing with the socio-economic level and appearing to be related to work status, MS, SC and income (p Conclusions Quality of life of Italians is still satisfactory, with a good degree of happiness and health, despite a clear decrease compared to 2004. The survey highlights that young people continue to perceive the future as uncertain, mainly due to job instability, with difficulty planning their lives. Key messages In ten years the Italian well-being has been reduced, despite the perceived level of happiness and health still being positive. The lack of job opportunities, the awareness of employment instability are the main causes of the lack of confidence in the future.

Background To understand the presence and impact of social and cultural factors on health vulnerability it is important for improving diabetes care and management. In fact, through the social dimension, it is possible to identify the priorities and attitudes towards diabetes and diabetes care among those living with the condition. Methods The study was carried out as part of the global Cities Changing Diabetes programme, involved a sample of individuals living with diabetes in the Rome metropolitan area and employed mixed-method and qualitative approaches to data collection and analysis (survey, sorting procedure and focus group). Results Four specific sub-groups of participants have been identified, each with distinct but shared priorities and attitudes towards diabetes: Health conscious thanks to the context; Medicalized elderly people; Fatalistic citizens; Worried but undisciplined young people. The connection between the place where you live and the possibility to adopt a healthier lifestyle was confirmed. For these patients, the disease is mainly characterized by its relationship with food and its connections with psychological aspects are also relevant. Conclusions An important issue concerns information and the different understandings of diabetes. A clear need emerged for further elaboration of the various aspects of a disease that tends to be underestimated also by those who have it. Another aspect concerns the importance of the living environment and consequently of the actions on its urban planning, mobility, but also in everyday life organization, as factors that can make a difference in properly managing the disease. These results are very important to promote a joint action, that have to involve public and private stakeholders, in order to improve treatment opportunities and quality of life of people facing diabetes every day in the Rome metropolitan area. Key messages An important issue concerns information. A clear need emerged for further elaboration of the various aspects of a disease that tends to be underestimated also by those who have it. The living environment in important too and the actions on its urban planning, mobility, in everyday life organization, as factors that can make a difference in properly managing the disease.

Background Multiple sclerosis (MS) is usually diagnosed between 20 and 40 years, affecting the most productive and active period of life. Aim To analyse the health perception of young people with MS and to compare the present results with a previous one. Methods A descriptive cross-sectional study was conducted in 2017. A validate and anonymous questionnaire was administered to 613 Italian young, aged 18-40, affected by MS (women 78.5%). Results Analyzing the declared health status, most of young MS defined good health status (76% good or excellent health) with differences between age class (82% at 25-29 years vs 71% at 35-40) and with a reverse trend with the increasing of disability (92% in subject with no disability vs 34% in ones with a medium or severe disability). The comparison against 2004 data highlights an improvement in the health conditions perceived, also for ones with a serious disability. Regarding the time elapsed from diagnosis, the trend was not linear, since respondents with a diagnosis Conclusions Diagnosis and disability constitute a decisive factor in the assessment of the state of health, but the perception goes to stabilize with time, even if the conditions tend to worsen. In this regard, the worst declared conditions in patients with a recent diagnosis seem to be influenced by psychological factors. So, the moment of diagnosis need a better management. Only 30% got adult vaccination (vs 61% in young Italians) due to lack of information on vaccine safety in MS patients. The improvement of perceived health in young with MS is another proof of the progress of MS treatments, which appears transversal to all the conditions of disability. Key messages The perception of health is strongly related to disability and to time elapsed from diagnosis. In the last 15 years the progress of MS management improved well-being of all MS young patients.

Background Public knowledge, awareness and behaviors related to antibiotic use and antimicrobial-resistance (AMR) are vital to ensure the prudent use of these drugs. In this work, we present the top-line results of the first national survey of AMR among the Italian general population aged ≥18 years. Methods From August to September 2019, we conducted a population-based cross-sectional random survey through mixed CATI/CAMI/CAWI methodologies among representative samples of the general population, parents of children aged 0-11 years, adults ≥65 years and pet's owners. Results were summarized using relative frequencies. Results Of 1,400 persons interviewed, 47% and 68% respectively correctly said that it is false that antibiotics kill viruses and are effective at treating colds and flu. More than 80% of respondents considered themselves to be well-informed about antibiotics but only one in two people knew what is meant by AMR. While the vast majority (96%) of respondents said that they obtained the last course of antibiotics that they used from a medical prescription, 11% incorrectly said that they stopped taking antibiotics when they felt better or that they used those left over from a previous course. In addition, 55% of pet's owners said they obtained antibiotics without a prescription from a pharmacy or elsewhere. Almost all respondents (97%) believed that it's everyone's responsibility to use antibiotics wisely, with the 80% who expressed concerns about the potential of AMR to affect themselves and their families; however, 40% did not think that action at an individual level is the most effective way of tackling resistance to antibiotics. Conclusions Our findings show some improvement in Italians' knowledge, awareness and behaviors related to antibiotic use and AMR but there are needs to be a greater sense of responsibility to take charge of the AMR problem. These results may be drivers of structured national awareness campaigns. Key messages In 2019, we conducted the first national survey of AMR among the Italian population: >80% of respondents felt themselves well-informed about antibiotics but only 1/2 people knew what is meant by AMR. Almost all Italian sample believed that it's everyone's responsibility to use antibiotics wisely, but 40% did not think that action at an individual level is effective to tackle AMR.

[Image: see text] Microsomal prostaglandin E(2) synthase-1 (mPGES-1), the terminal enzyme responsible for the production of inducible prostaglandin E(2), has become an attractive target for the treatment of inflammation and cancer pathologies. Starting from an aminobenzothiazole scaffold, used as an unprecedented chemical core for mPGES-1 inhibition, a Combinatorial Virtual Screening campaign was conducted, using the X-ray crystal structure of human mPGES-1. Two combinatorial libraries (6 × 10(4)) were obtained by decorating the aminobenzothiazole scaffold with all acyl chlorides and boronates available at the Merck database. The scientific multidisciplinary approach included virtual screening workflow, synthesis, and biological evaluation and led to the identification of three novel aminobenzothiazoles 1, 3, and 13 acting as mPGES-1 inhibitors. The three disclosed hits are able to inhibit mPGES-1 in a cell-free system (IC(50) = 1.4 ± 0.2, 0.7 ± 0.1, and 1.7 ± 0.2 μM, respectively), and all are endowed with antitumoral properties against A549 human cancer cell lines at micromolar concentrations (28.5 ± 1.1, 18.1 ± 0.8, and 19.2 ± 1.3 μM, respectively).

Heat shock protein 90 (Hsp90) is an ATP dependent molecular chaperone deeply involved in the complex network of cellular signaling governing some key functions, such as cell proliferation and survival, invasion and angiogenesis. Over the past years the N-terminal protein domain has been fully investigated as attractive strategy against cancer, but despite the many efforts lavished in the field, none of the N-terminal binders (termed “classical inhibitors”), currently in clinical trials, have yet successfully reached the market, because of the detrimental heat shock response (HSR) that showed to induce; thus, recently, the selective inhibition of Hsp90 C-terminal domain has powerfully emerged as a more promising alternative strategy for anti-cancer therapy, not eliciting this cell rescue cascade. However, the structural complexity of the target protein and, mostly, the lack of a co-crystal structure of C-terminal domain-ligand, essential to drive the identification of new hits, represent the largest hurdles in the development of new selective C-terminal inhibitors. Continuing our investigations on the identification of new anticancer drug candidates, by using an orthogonal screening approach, here we describe two new potent C-terminal inhibitors able to induce cancer cell death and a considerable down-regulation of Hsp90 client oncoproteins, without triggering the undesired heat shock response.

Cyclic peptoids have recently emerged as important examples of peptidomimetics for their interesting complexing properties and innate ability to permeate biological barriers. In the present contribution, experimental and theoretical data evidence the intricate conformational and stereochemical properties of five novel hexameric peptoids decorated with N-isopropyl, N-isobutyl, and N-benzyl substituents. Complexation studies by NMR, in the presence of sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (NaTFPB), theoretical calculations, and single-crystal X-ray analyses indicate that the conformationally stable host/guest metal adducts display architectural ordering comparable to that of the enniatins and beauvericin mycotoxins. Similarly to the natural depsipeptides, the synthetic oligolactam analogues show a correlation between ion transport abilities in artificial liposomes and cytotoxic activity on human cancer cell lines. The reported results demonstrate that the versatile cyclic peptoid scaffold, for its remarkable conformational and complexing properties, can morphologically mimic related natural products and elicit powerful biological activities.

Active flow control using fluidic actuators, via arrays of synthetic jet actuators, was used to provide control power for the Stingray unmanned aerial vehicle in the longitudinal (pitch) and lateral (roll) directions at low angles of attack. Using this technique, the pitch and roll moments were altered such that the effect is similar to that of a deflection of conventional control effectors. The control effectiveness of the synthetic jets on the aerodynamic performance of the Stingray unmanned aerial vehicle was investigated experimentally in a wind tunnel. Global flow measurements were conducted, where the moments and forces on the vehicle were measured using a six-component sting balance. The effect of the actuation was also examined on the surface static pressure at two spanwise locations. In addition, a particle image velocimetry technique was used to quantify the flowfield over the model, both the global flowfield as well as the localized interaction domain near the synthetic jet orifice. The synthetic jets were able to alter the local streamlines and displace the boundary layer through the formation of a small quasi-steady interaction region on the suction surface of the Stingray unmanned aerial vehicle's wing. Phase-locked particle image velocimetry data were acquired to provide insight into the growth, propagation, and decay of the synthetic jets impulse and their interaction with the crossflow. Furthermore, the changes induced on the moments and forces can be proportionally controlled by either changing the momentum coefficient or by driving the synthetic jets with a pulse modulation waveform. This can lead the way for future development of closed-loop control models.

Methylation in vivo is a post-translational modification observed in several organisms belonging to eucarya, bacteria, and archaea. Although important implications of this modification have been demonstrated in several eucaryotes, its biological role in hyperthermophilic archaea is far from being understood. The aim of this work is to clarify some effects of methylation on the properties of β-glycosidase from Sulfolobus solfataricus, by a structural comparison between the native, methylated protein and its unmethylated counterpart, recombinantly expressed in Escherichia coli. Analysis by Fourier transform infrared spectroscopy indicated similar secondary structure contents for the two forms of the protein. However, the study of temperature perturbation by Fourier transform infrared spectroscopy and turbidimetry evidenced denaturation and aggregation events more pronounced in recombinant than in native β-glycosidase. Red Nile fluorescence analysis revealed significant differences of surface hydrophobicity between the two forms of the protein. Unlike the native enzyme, which dissociated into SDS-resistant dimers upon exposure to the detergent, the recombinant enzyme partially dissociated into monomers. By electrospray mapping, the methylation sites of the native protein were identified. A computational analysis of β-glycosidase three-dimensional structure and comparisons with other proteins from S. solfataricus revealed analogies in the localization of methylation sites in terms of secondary structural elements and overall topology. These observations suggest a role for the methylation of lysyl residues, located in selected domains, in the thermal stabilization of β-glycosidase from S. solfataricus

The aim of this work is to provide insights into multiple metrics clinical validation of deformable image registration and contour propagation methods in 4D lung radiotherapy planning. The following indices were analyzed and compared: Volume Difference (VD), Dice Similarity Coefficient (DSC), Positive Predictive Value (PPV) and Surface Distances (SD). The analysis was performed on three patient datasets, using as reference a ground-truth volume generated by means of Simultaneous Truth And Performance Level Estimation (STAPLE) algorithm from the outlines of five experts. Significant discrepancies in the quality assessment provided by the different metrics in all the examined cases were found. Metrics sensitivity was more evident in presence of image artifacts and particularly for tubular anatomical structures, such as esophagus or spinal cord. Volume Differences did not account for position and DSC exhibited criticalities due to its intrinsic symmetry ( i.e. over- and under-estimation of the reference contours cannot be discriminated) and dependency on the total volume of the structure. PPV analysis showed more robust performance, as each voxel concurs to the classification of the propagation, but was not able to detect inclusion of propagated and ground-truth volumes. Mesh distances could interpret the actual shape of the structures, but might report higher mismatches in case of large local differences in the contour surfaces. According to our study, the combination of VD and SD for the validation of contour propagation algorithms in 4D could provide the necessary failure detection accuracy.

Besides its function in normal cellular growth, the molecular chaperone heat shock protein 90 (Hsp90) binds to a large number of client proteins required for promoting cancer cell growth and/or survival. In an effort to discover new small molecules able to inhibit the Hsp90 ATPase and chaperoning activities, we screened, by a surface plasmon resonance assay, a small library including different plant polyphenols. The ellagitannin geraniin, was identified as the most promising molecule, showing a binding affinity to Hsp90α similar to that of 17-(allylamino)-17-demethoxygeldanamycin (17AGG). Geraniin was able to inhibit in vitro the Hsp90α ATPase activity in a dose−dependent manner, with an inhibitory efficiency comparable to that measured for 17-AAG. In addition, this compound compromised the chaperone activity of Hsp90α, monitored by the citrate synthase thermal induced aggregation assay. Geraniin decreased the viability of HeLa and Jurkat cell lines and caused an arrest in G2/M phase. We also proved that following exposure to different concentrations of geraniin, the level of expression of the client proteins c-Raf, pAkt, and EGFR was strongly down−regulated in both the cell lines. These results, along with the finding that geraniin did not exert any appreciable cytotoxicity on normal cells, encourage further studies on this compound as a promising chemical scaffold for the design of new Hsp90 inhibitors.

The MSH2 c.388_389del mutation has occasionally been described in Lynch families worldwide. At the Portuguese Oncology Institute in Porto, Portugal, we have identified 16 seemingly unrelated families with this germline mutation. To evaluate if this alteration is a founder or a recurrent mutation we performed haplotype analysis in the 16 Portuguese index cases and 55 relatives, as well as in four index cases and 13 relatives reported from Germany, Scotland, England, and Argentina. In the Portuguese families we observed a shared haplotype of approximately 10 Mb and all were originated from the north of Portugal. These results suggest that this alteration is a founder mutation in Portugal with a relatively recent origin. In the reported families outside Portugal with this mutation different haplotype backgrounds were observed, supporting the hypothesis that it occurred de novo on multiple occasions. We also conclude that the high proportion of families with the MSH2 c.388_389del mutation indicates that screening for this alteration as a first step may be cost-effective in the genetic testing of Lynch syndrome suspects of Portuguese ancestry, especially those originating from the north of Portugal.

The category atypical glandular cells of undetermined significance (AGUS) occurs in about 0.5% of Papanicolaou smears. Although recent case series report a great many dysplastic, cancerous, and precancerous lesions of the cervix and endometrium associated with AGUS, little attention has been focused on this issue in primary care literature.We report a case series of 52 women with AGUS Papanicolaou smears in a family health center during 2 years (1997 to 1998), along with colposcopy and biopsy results and 18 months of follow-up findings. These results were compared with findings of recent reports.The incidence of AGUS was 0.5%, 52 of 10,564 Papanicolaou smears. Colposcopy was performed in 45. Biopsy (n = 46) showed 2 adenocarcinomas of the endometrium, and 6 high-grade dysplastic lesions, including 1 squamous cell carcinoma in situ. Nineteen women had cervical intraepithelial neoplasia stage I-changes of human papillomavirus effect, and 4 had endocervical polyps. Three women were lost to follow-up. The frequency of dysplastic and cancerous lesions in our population (17.4%) is consistent with series findings from cytology and obstetrics and gynecology literature, reporting that 19.5% of women with AGUS have either cancer--adenocarcinoma of the endometrium, squamous cell carcinoma, or extrauterine (8%)--or high-grade lesions--cervical intraepithelial neoplasia II-III, carcinoma in situ, or cervical adenocarcinoma in situ, (11.5%).A relatively large percentage of women with AGUS on Papanicolaou smears have cancerous and dysplastic squamous and glandular lesions of the exocervix, endocervix, and endometrium. Clinical practice guidelines recommend patients with AGUS should be evaluated with colposcopy and endocervical curettage. Consensus supports endometrial sampling in women 35 years and older and in those with a laboratory report of AGUS, favors neoplasia or suggests an endometrial source.

Postoperative irradiation of conservatively operated breast carcinoma is one of the most common practice in radiation oncology. In this job we analyze the acute toxicity during treatment.

The numerous demonstrations and disruptions occurring on this nation's campuses this past year have attracted the attention of many persons who, a decade ago, would not have known the difference between an ombudsman and "in loco parentis/' This nation-wide problem has grown from a single disturbance in 1964 to today's daily newspapers reporting campus disturbances as regularly as they do the closing stock averages. Clearly, public concern over our nation's colleges and universities has never been higher. Indeed, the growing number of campus disturbances has caused many persons to focus their attention on this enterprise called higher education, and to ask some searching questions about how colleges and universities are actually governed. In sorting out the possible responses to these questions, one finds less than total agreement on how decisions are made within academe, and more importantly, who (what persons or groups) ought rightfully to have a share in making them. In short, as we are beginning to realize, the real issue underlying many of our current campus problems is governance. And, when one raises the issue of governance, one is facing the fundamental political question; "Who decides what, where, when, and how?" To find answers to this question, one might begin, as have others with an analysis of the changing roles of students, faculty, administrators, and other influential who desire to direct today's colleges and universities by shaping academic policy. However, in approaching the problem in this manner, one faces the danger of being overly influenced by tradition, political pressures, economic sanctions, and many other considerations of a socio/politico nature. For example, we have until recently assumed that only those persons who have earned a college degree should be involved in the governance of institutions of higher education and the result is that our view of what students and/or faculty members are capable of achieving is highly distorted. To avoid this danger, I believe it advisable to begin by analyzing how

Hypotheses about the interactions of effectors with conformations of allosteric enzymes having co-operative kinetics can be tested simply and exactly without knowledge of the nature of the intersubunit co-operativity by using a linkage approach to the analysis of steady-state kinetics. Applying this approach to competition for substrate sites in the allosteric enzyme donkey spleen dCMP aminohydrolase, we show that the kinetics are consistent with the hypothesis that the substrate dCMP and the competitor dAMP, as well as the allosteric activator dCTP, bind exclusively to the same conformation of the enzyme subunits. The linkage test can be applied in the presence of other effectors without knowledge of how these interact with the enzyme. Our tests showed that dCMP and DAMP are still bound exclusively to this same conformation in the presence of the product dUMP or of the allosteric inhibitor dTTP. We give evidence that dUMP binds to the same conformation as dCMP, but that it is also bound to other conformation(s). The advantages of the linkage approach, and some general problems in steady-state kinetics of allosteric enzymes, are discussed.

In human red blood cells, protein 4.1 (4.1R) is an 80-kDa polypeptide that stabilizes the spectrin–actin network and anchors it to the plasma membrane. In non-erythroid cells there is a great variety of 4.1R isoforms, mainly generated by alternative pre-mRNA splicing, which localize at various intracellular sites, including the nucleus. We studied protein 4.1R distribution in relation to β-spectrin, actin and cytokeratin during Xenopus oogenesis. Immunoprecipitation experiments indicate that at least two isoforms of protein 4.1R are present in Xenopus laevis oocytes: a 56-kDa form in the cytoplasm and a 37-kDa form in the germinal vesicle (GV). Antibodies to β-spectrin reveal two bands of 239 and 100 kDa in the cytoplasm. Coimmunoprecipitation experiments indicate that both the 37- and 56-kDa isoforms of protein 4.1R associate with the 100-kDa isoform of β-spectrin. Moreover, the 56-kDa form coimmunoprecipitates with a cytokeratin of the same molecular weight. Confocal immunolocalization shows that protein 4.1R distribution is in the peripheral cytoplasm, in the mitochondrial cloud (MC) and in the GV of previtellogenic oocytes. In the cytoplasm of vitellogenic oocytes, a loose network of fibers stained by the anti-protein 4.1R antibody spreads across the cytoplasm. β-Spectrin has a similar distribution. Protein 4.1R was found to colocalize with actin in the cortex of oocytes in the form of fluorescent dots. Double immunolocalization of protein 4.1R and cytokeratin depicts two separate networks that overlap throughout the whole cytoplasm. Protein 4.1R filaments partially colocalize with cytokeratin in both the animal and vegetal hemispheres. We hypothesize that protein 4.1R could function as a linker protein between cytokeratin and the actin-based cytoskeleton.