Communication skills training for medical residents: Enhancing a psychosocial approach of patient care.

OBJECTIVES: One of the issues that has increasingly become relevant to medical practice is the ability to communicate well with patients. Better communication results in better care for the patient, as well as greater satisfaction for the physician. For this reason, the aim of this study was to assess the efficacy of a communication skills training program for medical residents (MR). METHOD: Eighty-six MR underwent a 6-month training program in three phases: a 12-h theory and practice workshop, a period of real practice, and a 4-h workshop in which the most challenging scenarios were role played with an actress. In each phase (T0, T1, and T2), participants' beliefs about their competence in caring for patients' psychosocial aspects and their self-confidence in communication skills were assessed. RESULTS: No differences were found between T0 and T1 in participants' beliefs of self-competence in psychosocial care. However, this competence significantly improved after completion of the entire program. Only 7 of the 12 areas explored in communication skills significantly improved between T0 and T1. However, after T2 completion, significant improvements were observed in all 12 areas. SIGNIFICANCE OF RESULTS: The research results highlight the usefulness and importance of training young doctors to foster their psychosocial approach to patient care and improve their confidence in their own communication skills. The results also show the appropriateness of the structure of the training: the key features of the programme were the follow-up of the participants in three phases over 6 months, and a focus on the needs of the residents and the resolution of difficult clinical cases, with the support of an actress. Therefore, the training presented in this study may become a guide for other trainings in other contexts with similar objectives.

Incorporation of 45S5 bioglass via sol-gel in ß-TCP scaffolds: Bioactivity and antimicrobial activity evaluation.

In this work, ß-TCP (ß-tricalcium phosphate) bioresorbable scaffolds were prepared by the gel casting method. Then, they were impregnated with a 45S5 bioglass sol gel solution to improve biocompatibility and promote bioactivity and antimicrobial activity. The ß-TCP scaffolds had an apparent porosity of 72%, and after the incorporation of the bioglass, this porosity was maintained. The elements of the bioglass were incorporated into ß-TCP matrix and there was a partial transformation from the ß-TCP phase to the α-TCP (α-tricalcium phosphate) phase, besides the formation of bioactive calcium and sodium­calcium silicates. The scaffolds ß-TCP with 45S5 bioglass incorporated (ß-TCP/45S5) did not show a reduction in their values of mechanical strength and Weibull modulus, despite the partial transformation to the α-TCP phase. Bioactivity, cell viability, and antimicrobial activity improved significantly for the ß-TCP/45S5 scaffold comparing to the scaffold without the bioglass. The mineralization of carbonated hydroxyapatite was verified in Simulated Body Fluid (SBF). The cell viability, evaluated by the reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide - MTT in MG63 cells, increased by 178%, and ß-TCP/45S5 scaffold also enhanced cell activity and osteoblast differentiation observed by means of total protein contend and alkaline phosphatase activity, respectively. The formation of growth inhibition zones was also observed in the disk diffusion assay for three tested microorganisms: Staphylococcus aureus, Escherichia coli and Candida albicans. To conclude, the vacuum impregnation method in 45S5 bioglass sol gel solution was effective in penetrating all the interconnected macroporosity of the scaffolds and covering the surface of the struts, which improved their biological properties in vitro, bioactivity and antibacterial activity, without reducing mechanical strength and porosity values. Thus, the ß-TCP/45S5 scaffolds are shown as potential candidates for use in tissue engineering, mainly in bone tissue regeneration and recovery.

Financial impact of healthcare-associated infections on intensive care units estimated for fifty Brazilian university hospitals affiliated to the unified health system.

BACKGROUND: Studies show that healthcare-associated infections (HAIs) represent a crucial issue in healthcare and can lead to substantial economic impacts in intensive care units (ICUs). AIM: To estimate direct costs associated with the most significant HAIs in 50 teaching hospitals in Brazil, affiliated to the unified health system (Sistema Único de Saúde: SUS). METHODS: A Monte Carlo simulation model was designed to estimate the direct costs of HAIs; first, epidemiologic and economic parameters were established for each HAI based on a cohort of 949 critical patients (800 without HAI and 149 with); second, simulation based on three Brazilian prevalence scenarios of HAIs in ICU patients (29.1%, 51.2%, and 61.6%) was used; and third, the annual direct costs of HAIs in 50 university hospitals were simulated. FINDINGS: Patients with HAIs had 16 additional days in the ICU, along with an extra direct cost of US$13.892, compared to those without HAIs. In one hypothetical scenario without HAI, the direct annual cost of hospital care for 26,649 inpatients in adult ICUs of 50 hospitals was US$112,924,421. There was an increase of approximately US$56 million in a scenario with 29.1%, and an increase of US$147 million in a scenario with 61.6%. The impact on the direct cost became significant starting at a 10% prevalence of HAIs, where US$2,824,817 is added for each 1% increase in prevalence. CONCLUSION: This analysis provides robust and updated estimates showing that HAI places a significant financial burden on the Brazilian healthcare system and contributes to a longer stay for inpatients.

Costs of healthcare-associated infections to the Brazilian public Unified Health System in a tertiary-care teaching hospital: a matched case-control study.

BACKGROUND: Little is known about the economic burden of healthcare-associated infections (HAIs) in Brazil. AIM: To analyse the costs of hospitalization by reimbursement from the Brazilian government, via the Brazilian Unified Health System (SUS) affiliation, and direct costs in the adult Intensive Care Unit (ICU). METHODS: The matched-pairs case-control study (83 patients with HAIs and 83 without HAIs) was performed at a referral tertiary-care teaching hospital in Brazil in January 2018. In order to calculate the HAI costs from the perspective of the payer, the total cost for each hospitalization was obtained through the Hospital's Billing Sector. Direct costs were calculated annually for 949 critical patients during 2018. FINDINGS: The reimbursement cost per hospitalization of patients with HAIs was 75% (US$2721) higher than patients without HAIs (US$1553). When a patient has an HAI, in addition to a longer length of stay (15 days), there was an extra increase (US$996) in the reimbursement cost per hospitalization. An HAI in the ICU was associated with a total direct cost eight times higher compared with patients who did not develop infections in this unit, US$11,776 × US$1329, respectively. The direct cost of hospitalization in the ICU without HAI was 56.5% less than the reimbursement (US$1329 × US$3052, respectively), whereas for the patient with an HAI, the direct cost was 111.5% above the reimbursement (US$11,776 × US$5569, respectively). CONCLUSION: HAIs contribute to a longer stay and an eight-fold increase in direct costs. It is necessary to reinforce programmes that prevent HAIs in Brazilian hospitals.

SAFIIRA: A heavy-ion multi-purpose irradiation facility in Brazil.

This work describes the new facility for applied nuclear physics at the University of Sao Paulo, mainly for irradiation of electronic devices. It is a setup composed of a quadrupole doublet for beam focusing/defocusing plus multiple scattering through gold foils to produce low intensity, large-area, and high-uniformity heavy-ion beams from 1H to 107Ag. Beam intensities can be easily adjusted from 102 particles cm2/s to hundreds of nA for an area as large as 2.0 cm2 and uniformity better than 90%. Its irradiation chamber has a high-precision motorized stage, and the system is controlled by a LabViewTM environment, allowing measurement automation. Design considerations and examples of use are presented.

Multi-hospital point prevalence study of healthcare-associated infections in 28 adult intensive care units in Brazil.

BACKGROUND: Healthcare-associated infection (HAI) represents a major problem for patient safety worldwide. AIM: To provide an up-to-date picture of the extent, aetiology, risk factors and patterns of infections in intensive care units (ICUs) in 28 Brazilian hospitals of different sizes. METHODS: A one-day point prevalence survey in 2016 enrolled the ICUs of hospitals from the 12 meso-regions in Minas Gerais state, south-east Brazil. Hospitals were classified as university or non-university hospitals. All patients with >48 h of admission to the study ICUs at the time of the survey were included. FINDINGS: In total, 303 patients were studied; of these, 155 (51.2%) were infected and 123 (79.4%) had at least one ICU-acquired infection. The most common ICU-acquired infections were pneumonia (53.0%) and bloodstream infection (27.6%). One hundred and nineteen bacterial isolates were cultured; the most common were Acinetobacter baumannii (27.1%), Pseudomonas aeruginosa (27.1%) and Staphylococcus aureus (39.0%). According to type of infection, the most common pathogens were P. aeruginosa (30.4%) in pneumonia, coagulase-negative staphylococci (23.4%) and Enterobacteriaceae (23.4%) in bloodstream infections, and Enterobacteriaceae (47.6%) in urinary tract infections. CONCLUSION: This study found that the overall prevalence of ICU-acquired infections in surveyed Brazilian hospitals was higher than that reported in most European countries and the USA. A greater proportion of infections were caused by non-fermenting Gram-negative bacteria. These observations, along with a high rate of antimicrobial use, illustrate the urgent need for HAIs to be a priority in the public health agenda of Brazil.

Diabetic foot ulcer development risk classifications' validation: A multicentre prospective cohort study.

AIMS: To prospectively validate the existing classifications to stratify subjects with diabetes mellitus (DM) by their risk of diabetic foot ulcer (DFU), in high and low risk settings. METHODS: A prospective multicentre cohort study was conducted, including 446 subjects with DM without active DFU followed in the hospital or primary care setting. Demographic, clinical characterization variables, and those included in the classifications were collected at baseline. Subjects were followed for 1year, until DFU or death. RESULTS: In our sample, with a mean age of 65years, 52% were male; 32 developed a DFU, 7 required an amputation and 18 died. Differences were found between participants' characteristics and classifications' accuracy according to the setting. The great majority of the variables were associated with higher DFU risk. Globally, classifications were highly and equally valid, positive predictive values (PV) were inferior to 40%, negative PV superior to 90% and area under the receiver operating characteristic curve superior to 0.75. DISCUSSION: All the existing classifications are valid to be applied in high risk clinical context and have a very high capacity to categorize as low risk those subjects that will not develop a DFU. Further research is needed in the primary care setting.

Brain volumetric and metabolic correlates of electroconvulsive therapy for treatment-resistant depression: a longitudinal neuroimaging study.

Recent research suggests that neuroplastic and neuroinflammatory changes may account for the mode of action of electroconvulsive therapy (ECT), although extant data do not allow for a clear disambiguation between these two hypotheses. Multimodal neuroimaging approaches (for example, combining structural and metabolic information) may help in clarifying this issue. Here we aimed to assess longitudinal changes in (i) regional gray matter (GM) volumes and (ii) hippocampal metabolite concentrations throughout an acute course of bitemporal ECT, as well as (iii) to determine the association between imaging changes and clinical improvement. We assessed 12 patients with treatment-resistant depression (TRD) at four time points (pre-treatment, after the first ECT session, after the ninth ECT session and 15 days after ECT course completion) and 10 healthy participants at two time points, 5 weeks apart. Patients with TRD showed bilateral medial temporal lobe (MTL) and perigenual anterior cingulate cortex volume increases. Left MTL volume increase was associated with (i) a hippocampal N-acetylaspartate concentration decrease, (ii) a hippocampal Glutamate+Glutamine concentration increase and (iii) significant clinical improvement. The observed findings are, in part, compatible with both neuroplastic and neuroinflammatory changes induced by ECT. We postulate that such phenomena may be interrelated, therefore reconciling the neuroplasticity and neuroinflammatory hypotheses of ECT action.

Cross-cultural measurement invariance of the Revised Child Anxiety and Depression Scale across 11 world-wide societies.

BACKGROUND: In order to compare estimates by one assessment scale across various cultures/ethnic groups, an important aspect that needs to be demonstrated is that its construct across these groups is invariant when measured using a similar and simultaneous approach (i.e., demonstrated cross-cultural measurement invariance). One of the methods for evaluating measurement invariance is testing for differential item functioning (DIF), which assesses whether different groups respond differently to particular items. The aim of this study was to evaluate the cross-cultural measurement invariance of the Revised Child Anxiety and Depression Scale (RCADS) in societies with different socioeconomic, cultural, and religious backgrounds. METHODS: The study was organised by the International Child Mental Health Study Group. Self-reported data were collected from adolescents residing in 11 countries: Brazil, Bulgaria, Croatia, Indonesia, Montenegro, Nigeria, Palestinian Territories, the Philippines, Portugal, Romania and Serbia. The multiple-indicators multiple-causes model was used to test the RCADS items for DIF across the countries. RESULTS: Ten items exhibited DIF considering all cross-country comparisons. Only one or two items were flagged with DIF in the head-to-head comparisons, while there were three to five items flagged with DIF, when one country was compared with the others. Even with all cross-culturally non-invariant items removed from nine language versions tested, the original factor model representing six anxiety and depressive symptoms subscales was not significantly violated. CONCLUSIONS: There is clear evidence that relatively small number of the RCADS items is non-invariant, especially when comparing two different cultural/ethnic groups, which indicates on its sound cross-cultural validity and suitability for cross-cultural comparisons in adolescent anxiety and depressive symptoms.

Outbreaks of colistin-resistant and colistin-susceptible KPC-producing Klebsiella pneumoniae in a Brazilian intensive care unit.

BACKGROUND: Carbapenem-resistant Enterobacteriaceae (CRE), especially those that produce Klebsiella pneumoniae carbapenemase (KPC) and are associated with colistin resistance, pose a severe health threat due to the limited treatment options. AIM: To describe two outbreaks of KPC-producing K. pneumoniae in an adult intensive care unit (AICU) in Brazil. In May 2015, 14 patients had colistin-susceptible KPC-producing strains (ColS-KPC), and in July 2015, nine patients had colistin-resistant KPC-producing strains (ColR-KPC). METHODS: Between September 2014 and August 2015, we performed surveillance at a university hospital and all CRE were tested for blaKPC genes. Clonality was investigated by pulsed-field gel electrophoresis. Resistance to colistin was confirmed by broth microdilution method. Consumption of carbapenems and colistin was expressed as defined daily doses. FINDINGS: In all, 111 patients with CRE were identified during the surveillance period; K. pneumoniae was the major isolate (77.13%). The two outbreaks were identified when infection rates (KPC per 1000 patient-days) exceeded the background level. Rates of carbapenem and colistin consumption were high. Control measures (bedside alcohol gel, contact precautions, regular rectal swabs) did not curtail the outbreaks. Mortality rates were 42.9% and 44.4% for ColS-KPC- and ColR-KPC-infected patients, respectively. After the death of four infected patients with ColR-KPC, the unit was closed to new admissions. CONCLUSION: Our experience demonstrates the serious risks presented by KPC, and especially ColR-KPC, in Brazilian AICUs. Selective pressure from excessive antibiotic use and transmission on healthcare workers' hands were likely the major factors in transmission.

Attitudes and intentions to smoke: a study of young Brazilian children.

BACKGROUND: Children at earlier stages tend to be more susceptible towards different types of tobacco messages. These are able to influence attitudes and behaviours around smoking. This study examined how these messages are able to influence 5- and 6-year olds' attitudes about smokers and their smoking intentions. METHODS: Researchers worked one-on-one with 5- and 6-year olds (n = 398) in Brazil. Children could attribute positive and negative characteristics to two different persons in photos as smoker/non-smoker. Children could indicate the attribute as of a smoker, a non-smoker, both or none. Children were asked also about their smoking intentions. Analysis considered parental smoking, sex, age, cigarette and alcohol brand logos, children's location and media characters from cartoons. We conducted a path analysis for a multivariate model of children's attitudes and intentions about smoking. RESULTS: Overall, children had negative attitudes about smokers (M = -4.58, SD = 4.08) and a total of 32 (8.0%) of them reported intentions to smoke. The resulting multivariate model indicates that parental smoking is a source for a positive image of smokers, while being 6 years old, living in rural areas, being aware of alcohol brands and recognizing educational cartoons tended to be negatively correlated to children's attitudes. Further, 6 year olds were found to be less likely to have smoking intentions, while attitude about smokers was positively related with intentions. One's attitudes served as a mediator for all of the variables in the model towards smoking intentions. The path models differed for each sex. CONCLUSION: Shaped by social and environmental influences, very young children have opinions about smokers. In turn, these attitudes significantly predict children's smoking intentions. To corroborate this research, we recommend that longitudinal designs be employed to help model why Brazilian children become smokers.

A system to measure isomeric state half-lives in the 10 ns to 10 µs range.

The Isomeric State Measurement System (SISMEI) was developed to search for isomeric nuclear states produced by fusion-evaporation reactions. The SISMEI consists of 10 plastic phoswich telescopes, two lead shields, one NaI(Tl) scintillation detector, two Compton suppressed HPGe γ-ray detectors, and a cone with a recoil product catcher. The new system was tested at the 8 UD Pelletron tandem accelerator of the University of São Paulo with the measurement of two known isomeric states: (54)Fe, 10(+) state (E = 6527.1 (11) keV, T(1/2) = 364(7) ns) and the 5/2(+) state of (19)F (E = 197.143 (4) keV, T(1/2) = 89.3 (10) ns). The results indicate that the system is capable of identifying delayed transitions, of measuring isomeric state lifetimes, and of identifying the feeding transitions of the isomeric state through the delayed γ-γ coincidence method. The measured half-life for the 10(+) state was T(1/2) = 365(14) ns and for the 5/2(+) state, 100(36) ns.

Bacterial colonization of pressure ulcers: assessment of risk for bloodstream infection and impact on patient outcomes.

BACKGROUND: Pressure ulcers (PUs) represent a major problem for hospitalized patients, health professionals and society. AIM: To evaluate the impact of in-hospital PUs as a reservoir of multidrug-resistant organisms (MDROs), risk factor for bacteraemia and predictor of poor prognosis. METHODS: A prospective cohort study of patients with stage II or greater PUs hospitalized in a tertiary acute care university teaching hospital for more than 48 h was performed to evaluate colonization/infection by potential and/or multi-resistant hospital pathogens from April to December 2005 and from August 2009 to April 2010. FINDINGS: A total of 145 patients with stage II or greater PUs were included. Of these, 76.5% (111/145) had PUs colonized and/or infected with either S. aureus (20.7%), Gram-negative bacilli (32.5%), or both (46.8%) and most were MDROs (64.8%). Bacteraemia was detected in 50.5% (56/111) of the patients. The ulcers were considered to be the probable source of bacteraemia in 53.6% (30/56) of the episodes. Prior administration of antibiotics (P = 0.04) and infected wound (P < 0.001) were the variables independently associated with bloodstream infection as well as associated with a higher 30-day mortality rate; risk factors for the latter included hospitalization in ICU (P = 0.03) and mechanical ventilation use (P = 0.05). CONCLUSIONS: Our results suggest that besides being a major reservoir of MDROs, patients with PUs constitute a high-risk population for bacteraemia with a poor outcome. Broad-spectrum antibiotics and infected wound were independent factors predisposing patients to both bacteraemia and death.

Structural covariance of the neostriatum with regional gray matter volumes.

The caudate and putamen nuclei have been traditionally divided into dorsal and ventral territories based on their segregated patterns of functional and anatomical connectivity with distributed cortical regions. Activity-dependent structural plasticity may potentially lead to the development of regional volume correlations, or structural covariance, between the different components of each cortico-striatal circuit. Here, we studied the whole-brain structural covariance patterns of four neostriatal regions belonging to distinct cortico-striatal circuits. We also assessed the potential modulating influence of laterality, age and gender. T1-weighted three-dimensional magnetic resonance images were obtained from ninety healthy participants (50 females). Following data pre-processing, the mean signal value per hemisphere was calculated for the 'seed' regions of interest, located in the dorsal and ventral caudate and the dorsal-caudal and ventral-rostral putamen. Statistical parametric mapping was used to estimate whole-brain voxel-wise structural covariance patterns for each striatal region, controlling for the shared anatomical variance between regions in order to obtain maximally specific structural covariance patterns. As predicted, segregated covariance patterns were observed. Age was found to be a relevant modulator of the covariance patterns of the right caudate regions, while laterality effects were observed for the dorsal-caudal putamen. Gender effects were only observed via an interaction with age. The different patterns of structural covariance are discussed in detail, as well as their similarities with the functional and anatomical connectivity patterns reported for the same striatal regions in other studies. Finally, the potential mechanisms underpinning the phenomenon of volume correlations between distant cortico-striatal structures are also discussed.

A large Venous-Arterial PCO(2) Is Associated with Poor Outcomes in Surgical Patients.

Background. This study evaluated whether large venous-arterial CO(2) gap (PCO(2) gap) preoperatively is associated to poor outcome. Method. Prospective study which included adult high-risk surgical patients. The patients were pooled into two groups: wide [P(v-a)CO(2)] versus narrow [P(v-a)CO(2)]. In order to determine the best value to discriminate hospital mortality, it was applied a ROC (receiver operating characteristic) curve for the [P(v-a)CO(2)] values collected preoperatively, and the most accurate value was chosen as cut-off to define the groups. Results. The study included 66 patients. The [P(v-a)CO(2)] value preoperatively that best discriminated hospital mortality was 5.0 mmHg, area = 0.73. Preoperative patients with [P(v-a)CO(2)] more than 5.0 mmHg presented a higher hospital mortality (36.4% versus 4.5% P = 0.004), higher prevalence of circulatory shock (56.8% versus 22.7% P = 0.01) and acute renal failure postoperatively (27.3% versus 4.5% P = 0.02), and longer hospital length of stays 20.0 (14.0-30.0) versus 13.5 (9.0-25.0) days P = 0.01. Conclusions. The PCO(2) gap values more than 5.0 mmHg preoperatively were associated with worse postoperatively outcome.

Purification and partial characterization of a bacteriocin produced by an oral Fusobacterium nucleatum isolate.

AIMS: To purify and partially characterize a bacteriocin produced by a Fusobacterium nucleatum strain. METHODS AND RESULTS: Following protein precipitation the effect of different treatments on a bacteriocin produced by a F. nucleatum strain named P12.2 isolated from a patient with periodontitis was evaluated. The antagonistic activity of the intracellular fraction obtained at 80% ammonium sulphate was preserved at pH values from 6.0 to 9.0 and showed to be sensitive to high temperatures and to treatment with proteases. The fraction was submitted to sequential steps of gel filtration, ion exchange, and reverse phase chromatography, and SDS-PAGE. Data obtained by mass spectrometry revealed that the molecular mass of the protein was 27,296 Da. CONCLUSIONS: For the first time a bacteriocin produced by a F. nucleatum strain was purified and characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first description on characterization of a bacteriocin produced by F. nucleatum. It is possible that the bacteriocin plays a role in the regulation of population levels of periodontopathic organisms.

Production of antagonistic substance by Eikenella corrodens isolated from the oral cavity of human beings with and without periodontal disease.

AIMS: Antagonistic abilities may confer ecological advantages for micro-organisms in competitive ecosystems. However, reports regarding this phenomenon in Eikenella corrodens are not available. METHODS AND RESULTS: Nineteen E. corrodens strains, isolated from the oral cavity of human beings without periodontal disease (n = 5) and with aggressive (n = 9) and chronic (n = 5) periodontitis, as well as a reference strain (E. corrodens ATCC23834), were evaluated for antagonistic activity. The following indicators were used: Porphyromonas gingivalis FDC381, Prevotella intermedia ATCC25611, Actinomyces israelii ATCC12102, Eubacterium lentum ATCC25559, Peptostreptococcus anaerobius ATCC27337, Actinobacillus actinomycetemcomitans FDCY4, Fusobacterium nucleatum ATCC10953, Streptococcus sanguinis ATCC10557, Streptococcus uberis ATCC9927, Streptococcus mutans IM/UFRJ, Staphylococcus aureus ATCC33591 and Candida albicans ATCC18804. All the strains showed antagonism against at least one of the indicator strains. This phenomenon was more frequently observed for strains isolated from patients with chronic periodontitis (36.4%), than those from healthy subjects (20.6%) and those with aggressive periodontitis (10.8%). CONCLUSIONS: The heterogeneous antagonistic spectrum exhibited by E. corrodens isolates suggests their ability to produce more than one antagonistic substance, whose ecological relevance is yet to be demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first description of antagonistic compound production by E. corrodens and its relationships with the clinical status of the patients.

Modifications to improve the efficiency of zona-free mouse nuclear transfer.

In the present study, some modifications were made to the zona-free nuclear transfer technique in the mouse in order to achieve greater efficiency. Firstly, a 1-h interval was allowed between cumulus removal and zona pellucida digestion. Secondly, acid Tyrode's was selected for zona pellucida removal, because contrary to pronase, it allows embryo survival during parthenogenic activation in the absence of calcium. Even when the exposure time to pronase was reduced to as little as 1 min or washed with fetal calf serum to inhibit the enzyme, the percentage of lysis during activation in the absence of calcium was still very high. Thirdly, electrofusion was performed at room temperature (21 degrees C), instead of 30 degrees C as in our previous experiments. Finally, embryos were cultured in groups of 12-15, instead of individually, using a "well of the wells" system during activation and culture. When compared, parthenogenic activated control embryos showed an increase in the development to blastocyst when cultured in pairs instead of individually. By the end of the experiments and using embryonic stem (ES) cells, there was a significant increase in fusion rate (1.5-fold increase) and in development to morula/blastocyst from cleaved reconstructed embryos (1.5-fold increase) when compared with the results before the modifications. A 2.4-fold increase in overall efficiency was achieved from the oocyte to morula/blastocyst stages.

Development of a zona-free method of nuclear transfer in the mouse.

In the present study, a zona-free nuclear transfer (NT) technique, which had been originally developed in cattle, was modified for the mouse. Steps involved in this approach include removing the zona pellucida and enucleating without a holding pipette; sticking donor cells to the cytoplast before electric pulses are applied to fuse them and culturing reconstructed embryos individually in single droplets, to prevent aggregation. Control zona-free and zona-intact embryos from mated donors showed no significant difference in development to blastocyst, but did show reduced development to term. Removal of the zona pellucida affected the response to activation by strontium in the absence of calcium as a significant proportion of zona-free control oocytes and embryos reconstructed by NT lysed during this treatment. A comparison between cumulus and ES cells as donor cells revealed significant differences in fusion efficiency (58.1 +/- 4.0%, n = 573 vs. 42.9 +/- 2.2%, n = 2064, respectively, p < 0.001), cleavage (77.2 +/- 3.4%, n = 334 vs. 40.8 +/- 2.7%, n = 903, respectively, p < 0.001) but not for development to morula/blastocyst (8.7 +/- 2.1%, n = 334 vs. 13.9 +/- 1.8%, n = 903, respectively, p < 0.1). The stage at which embryo development arrested was also affected by donor cell type. A majority of embryos reconstructed from cumulus cells arrested at two-cell stage, usually with two nuclei, whereas those reconstructed from ES cells arrested at one-cell stage, usually with two pseudo-pronuclei. After transfer of ES cell-derived NT embryos, a viable cloned mouse was produced (3.0% of transferred embryos developed to term). These observations establish that a zona-free cloning approach is possible in the mouse, although further research is required to increase the efficiency.