Influence of Deep Sedation in Intensive Care Medicine Memories of Critical COVID-19 Survivors.

Introduction: Critical care survivors sustain a variety of sequelae after intensive care medicine (ICM) admission, and the Coronavirus Disease 2019 (COVID-19) pandemic has added further challenges. Specifically, ICM memories play a significant role, and delusional memories are associated with poor outcomes post-discharge including a delayed return to work and sleep problems. Deep sedation has been associated with a greater risk of perceiving delusional memories, bringing a move toward lighter sedation. However, there are limited reports on post-ICM memories in COVID-19, and influence of deep sedation has not been fully defined. Therefore, we aimed to evaluate ICM-memory recall in COVID-19 survivors and their relation with deep sedation. Materials/Methods: Adult COVID-19 ICM survivors admitted to a Portuguese University Hospital between October 2020 and April 2021 (second/third "waves") were evaluated 1 to 2 months post-discharge using "ICU Memory Tool," to assess real, emotional, and delusional memories. Results: The study included 132 patients (67% male; median age = 62 years, Acute Physiology and Chronic Health Evaluation [APACHE]-II = 15, Simplified Acute Physiology Score [SAPS]-II = 35, ICM stay = 9 days). Approximately 42% received deep sedation (median duration = 19 days). Most participants reported real (87%) and emotional (77%) recalls, with lesser delusional memories (36.4%). Deeply sedated patients reported significantly fewer real memories (78.6% vs 93.4%, P = .012) and increased delusional memories (60.7% vs 18.4%, P < .001), with no difference in emotional memories (75% vs 80.4%, P = .468). In multivariate analysis, deep sedation had a significant, independent association with delusional memories, increasing their likelihood by a factor of approximately 6 (OR = 6.274; 95% confidence interval = 1.165-33.773, P = .032), without influencing real (P = .545) or emotional (P = .133) memories. Conclusions: This study contributes to a better understanding of the potential adverse effects of deep sedation on ICM memories in critical COVID-19 survivors, indicating a significant, independent association with the incidence of delusional recalls. Although further studies are needed to support these findings, they suggest that strategies targeted to minimize sedation should be favored, aiming to improve long-term recovery.

ESMO Expert Consensus Statements on Cancer Survivorship: promoting high-quality survivorship care and research in Europe.

BACKGROUND: The increased number of cancer survivors and the recognition of physical and psychosocial challenges, present from cancer diagnosis through active treatment and beyond, led to the discipline of cancer survivorship. DESIGN AND METHODS: Herein, we reflected on the different components of survivorship care, existing models and priorities, in order to facilitate the promotion of high-quality European survivorship care and research. RESULTS: We identified five main components of survivorship care: (i) physical effects of cancer and chronic medical conditions; (ii) psychological effects of cancer; (iii) social, work and financial effects of cancer; (iv) surveillance for recurrences and second cancers; and (v) cancer prevention and overall health and well-being promotion. Survivorship care can be delivered by structured care models including but not limited to shared models integrating primary care and oncology services. The choice of the care model to be implemented has to be adapted to local realities. High-quality care should be expedited by the generation of: (i) focused and shared European recommendations, (ii) creation of tools to facilitate implementation of coordinated care and (iii) survivorship educational programs for health care teams and patients. The research agenda should be defined with the participation of health care providers, researchers, policy makers, patients and caregivers. The following patient-centered survivorship research areas were highlighted: (i) generation of a big data platform to collect long-term real-world data in survivors and healthy controls to (a) understand the resources, needs and preferences of patients with cancer, and (b) understand biological determinants of survivorship issues, and (ii) develop innovative effective interventions focused on the main components of survivorship care. CONCLUSIONS: The European Society for Medical Oncology (ESMO) can actively contribute in the efforts of the oncology community toward (a) promoting the development of high-quality survivorship care programs, (b) providing educational material and (c) aiding groundbreaking research by reflecting on priorities and by supporting research networking.

Supporting the gastrointestinal microenvironment during high-dose chemotherapy and stem cell transplantation by inhibiting IL-1 signaling with anakinra.

High-dose chemotherapy causes intestinal inflammation and subsequent breakdown of the mucosal barrier, permitting translocation of enteric pathogens, clinically manifesting as fever. Antibiotics are mainstay for controlling these complications, however, they are increasingly recognized for their detrimental effects, including antimicrobial resistance and dysbiosis. Here, we show that mucosal barrier injury induced by the mucotoxic chemotherapeutic agent, high-dose melphalan (HDM), is characterized by hyper-active IL-1b/CXCL1/neutrophil signaling. Inhibition of this pathway with IL-1RA, anakinra, minimized the duration and intensity of mucosal barrier injury and accompanying clinical symptoms, including diarrhea, weight loss and fever in rats. 16S analysis of fecal microbiome demonstrated a more stable composition in rats receiving anakinra, with reduced pathogen expansion. In parallel, we report through Phase IIA investigation that anakinra is safe in stem cell transplant patients with multiple myeloma after HDM. Ramping-up anakinra (100-300 mg administered intravenously for 15 days) did not cause any adverse events or dose limiting toxicities, nor did it change time to neutrophil recovery. Our results reinforce that strengthening the mucosal barrier may be an effective supportive care strategy to mitigate local and systemic clinical consequences of HDM. We are now conducting a Phase IIB multicenter, placebo-controlled, double-blinded trial to assess clinical efficacy of anakinra (AFFECT-2).Trial registration: ClinicalTrials.gov identifier: NCT03233776.

Development of a self-limiting model of methotrexate-induced mucositis reinforces butyrate as a potential therapy.

Gastrointestinal mucositis is a complication of anticancer treatment, with few validated in vitro systems suitable to study the complex mechanisms of mucosal injury. Therefore, we aimed to develop and characterize a chemotherapeutic-induced model of mucositis using 3D intestinal organoids. Organoids derived from mouse ileum were grown for 7 days and incubated with different concentrations of the chemotherapeutic agent methotrexate (MTX). Metabolic activity, citrulline levels and cytokine/chemokine production were measured to determine the optimal dosage and incubation time. The protective effects of folinic acid on the toxicity of MTX were investigated by pre-treating organoids with (0.0005-50 µg/mL) folinic acid. The impact of microbial-derived short-chain fatty acids was evaluated by supplementation with butyrate in the organoid model. MTX caused a dose-dependent reduction in cell metabolic activity and citrulline production that was salvaged by folinic acid treatment. Overall, MTX causes significant organoid damage, which can be reversed upon removal of MTX. The protective effect of folinic acid suggest that the organoids respond in a clinical relevant manner. By using the model for intervention, it was found that prophylactic treatment with butyrate might be a valuable strategy for prophylactic mucositis prevention.

Safety of assisted reproductive techniques in young women harboring germline pathogenic variants in BRCA1/2 with a pregnancy after prior history of breast cancer.

BACKGROUND: Knowledge is growing on the safety of assisted reproductive techniques (ART) in cancer survivors. No data exist, however, for the specific population of breast cancer patients harboring germline BRCA1/2 pathogenic variants. PATIENTS AND METHODS: This is a multicenter retrospective cohort study across 30 centers worldwide including women diagnosed at ≤40 years with stage I-III breast cancer, between January 2000 and December 2012, harboring known germline BRCA1/2 pathogenic variants. Patients included in this analysis had a post-treatment pregnancy either achieved through use of ART (ART group) or naturally (non-ART group). ART procedures included ovulation induction, ovarian stimulation for in vitro fertilization or intracytoplasmic sperm injection, and embryo transfer under hormonal replacement therapy. RESULTS: Among the 1424 patients registered in the study, 168 were eligible for inclusion in the present analysis, of whom 22 were in the ART group and 146 in the non-ART group. Survivors in the ART group conceived at an older age compared with those in the non-ART group (median age: 39.7 versus 35.4 years, respectively). Women in the ART group experienced more delivery complications compared with those in the non-ART group (22.1% versus 4.1%, respectively). No other apparent differences in obstetrical outcomes were observed between cohorts. The median follow-up from pregnancy was 3.4 years (range: 0.8-8.6 years) in the ART group and 5.0 years (range: 0.8-17.6 years) in the non-ART group. Two patients (9.1%) in the ART group experienced a disease-free survival event (specifically, a locoregional recurrence) compared with 40 patients (27.4%) in the non-ART group. In the ART group, no patients deceased compared with 10 patients (6.9%) in the non-ART group. CONCLUSION: This study provides encouraging safety data on the use of ART in breast cancer survivors harboring germline pathogenic variants in BRCA1/2, when natural conception fails or when they opt for ART in order to carry out preimplantation genetic testing.

Translational model of melphalan-induced gut toxicity reveals drug-host-microbe interactions that drive tissue injury and fever.

PURPOSE: Conditioning therapy with high-dose melphalan (HDM) is associated with a high risk of gut toxicity, fever and infections in haematopoietic stem cell transplant (HSCT) recipients. However, validated preclinical models that adequately reflect clinical features of melphalan-induced toxicity are not available. We therefore aimed to develop a novel preclinical model of melphalan-induced toxicity that reflected well-defined clinical dynamics, as well as to identify targetable mechanisms that drive intestinal injury. METHODS: Male Wistar rats were treated with 4-8 mg/kg melphalan intravenously. The primary endpoint was plasma citrulline. Secondary endpoints included survival, weight loss, diarrhea, food/water intake, histopathology, body temperature, microbiota composition (16S sequencing) and bacterial translocation. RESULTS: Melphalan 5 mg/kg caused self-limiting intestinal injury, severe neutropenia and fever while impairing the microbial metabolome, prompting expansion of enteric pathogens. Intestinal inflammation was characterized by infiltration of polymorphic nuclear cells in the acute phases of mucosal injury, driving derangement of intestinal architecture. Ileal atrophy prevented bile acid reabsorption, exacerbating colonic injury via microbiota-dependent mechanisms. CONCLUSION: We developed a novel translational model of melphalan-induced toxicity, which has excellent homology with the well-known clinical features of HDM transplantation. Application of this model will accelerate fundamental and translational study of melphalan-induced toxicity, with the clinical parallels of this model ensuring a greater likelihood of clinical success.

Pre-therapy fasting slows epithelial turnover and modulates the microbiota but fails to mitigate methotrexate-induced gastrointestinal mucositis.

BACKGROUND: Recent findings by Tang et al. (2020) show dietary restriction (30%, 2 weeks) prevents methotrexate-induced mortality by modulation of the microbiota, specifically the expansion of Lactobacillus. While fundamentally insightful, upscaling this schedule is a major obstacle to clinical uptake. Here, we evaluate a safe and clinically achievable schedule of pre-therapy fasting for 48 h on microbiota composition, body composition and intestinal proliferation, and assess its impact on the severity of methotrexate-induced gastrointestinal mucositis using a validated preclinical rat model. METHODS: Age- and weight-matched male Wistar rats were treated with a sublethal dose of 45 mg/kg methotrexate with or without pre-therapy fasting. The impact of acute fasting on epithelial proliferation, body composition and the microbiota was assessed using plasma citrulline, Ki67 immunohistochemistry, miniSpec and 16S rRNA sequencing. The severity of gastrointestinal mucositis was evaluated using plasma citrulline and body weight. RESULTS: Whilst pre-therapy fasting slowed epithelial proliferation and increased microbial diversity and richness, it also induced significant weight loss and was unable to attenuate the severity of mucositis in both age- and weight-matched groups. In contrast to Tang et al., we saw no expansion of Lactobacillus following acute fasting. CONCLUSIONS: Our findings suggest that the beneficial effects of acute fasting are masked by the detrimental effects on body weight and composition and lacking influence on Lactobacillus. Future studies should consider alternative fasting schedules or aim to induce comparable microbial and mucosal manipulation without compromising body composition using clinically feasible methods of dietary or microbial intervention.

Differential impact of endocrine therapy and chemotherapy on quality of life of breast cancer survivors: a prospective patient-reported outcomes analysis.

BACKGROUND: In early breast cancer (BC), there has been a trend to escalate endocrine therapy (ET) and to de-escalate chemotherapy (CT). However, the impact of ET versus CT on the quality of life (QoL) of early BC patients is unknown. Here, we characterize the independent contribution of ET and CT on patient-reported outcomes (PROs) at 2 years after diagnosis. PATIENTS AND METHODS: We prospectively collected PROs in 4262 eligible patients using the European Organization for Research and Treatment of Cancer QLQ-C30/BR23 questionnaires inside CANTO trial (NCT01993498). The primary outcome was the C30 summary score (C30-SumSc) at 2 years after diagnosis. RESULTS: From eligible patients, 37.2% were premenopausal and 62.8% postmenopausal; 81.9% received ET and 52.8% CT. In the overall cohort, QoL worsened by 2 years after diagnosis in multiple functions and symptoms; exceptions included emotional function and future perspective, which improved over time. ET (Pint = 0.004), but not CT (Pint = 0.924), had a persistent negative impact on the C30-SumSc. In addition, ET negatively impacted role and social function, pain, insomnia, systemic therapy side-effects, breast symptoms and further limited emotional function and future perspective recovery. Although CT had no impact on the C30-SumSc at 2-years it was associated with deteriorated physical and cognitive function, dyspnea, financial difficulties, body image and breast symptoms. We found a differential effect of treatment by menopausal status; in premenopausal patients, CT, despite only a non-significant trend for deteriorated C30-SumSc (Pint = 0.100), was more frequently associated with QoL domains deterioration than ET, whereas in postmenopausal patients, ET was more frequently associated with QoL deterioration, namely using the C30-SumSc (Pint = 0.004). CONCLUSION(S): QoL deterioration persisted at 2 years after diagnosis with different trajectories by treatment received. ET, but not CT, had a major detrimental impact on C30-SumSc, especially in postmenopausal women. These findings highlight the need to properly select patients for adjuvant ET escalation.

Serological evidence of Borrelia circulation among blood donors in the São Paulo state, Brazil.

OBJECTIVE: The objective of this study was to examine the Borrelia seroprevalence among blood donors in Southeast Brazil. BACKGROUND: There is evidence that Borrelia spirochetes are circulating in Brazil; however, there are no studies that characterise these bacteria and investigate their seroprevalence in the Brazilian population. Such a situation, combined with a recent outbreak of tick-borne Rocky Mountain spotted fever in the São Paulo state demonstrates the increasing role of ticks as arthropod vectors in Brazil. METHODS: For the purpose of the study, 452 blood donors from Ribeirão Preto city, São Paulo state were tested using anti-Borrelia immunoglobulin G (IgG) assay. The positive results were also confirmed by Western blot for anti-borrelia IgM/IgG. RESULTS: The anti-Borrelia IgG enzyme-linked immunosorbent assay (ELISA) showed nine positive and nine borderline reactive samples, giving a total seroprevalence of 2·0% of anti-Borrelia IgG among Brazilian blood donors. The confirmation of the seropositive and borderline samples by Borrelia Western blot was demonstrated by IgG-positive results in 16 samples (a seroprevalence of 3.5%). Anti-Borrelia IgM antibodies were also detected in one sample. CONCLUSIONS: Our results demonstrate that Borrelia-like spirochetes may be circulating among blood donors from the São Paulo State and that the ticks have an important epidemiological role as vectors of bacterial infections in this Brazilian region. These results not only alert us to possible actions that might be undertaken in order to completely characterise the aetiological agents of Lyme-like syndromes in Brazil but also the possible impact that these bacterial agents might have on haemotherapy practices.

A partial least squares - Path modeling analysis for the understanding of biodiversity loss in rural and urban watersheds in Portugal.

The main purpose of this study was to use Partial Least Squares - Path Modeling (PLS-PM) to quantify the contributions of natural and human-induced threats to biodiversity loss in rural and urban watersheds. The study area comprised the Sabor and Ave river basins, located in northern Portugal. The Sabor is rural and sparsely populated while the Ave is urbanized, industrialized and densely populated. Within PLS-PM, threats are called exogenous latent variables while the ultimate environmental consequence (biodiversity loss) is termed endogenous latent variable. Latent variables are concepts represented by numerical parameters called formative variables. The selected latent variables were given the names "pressures", "contamination" and "ecological integrity". The most important "pressures" were the wildfire risk, the percentage of urban area in sub-catchments, the diffuse emissions of livestock nitrogen (N) and agriculture/forest phosphorus (P), and the point source emissions of urban N, P and biochemical oxygen demand, as well as of industrial N. The latent variable called "contamination" was primarily represented by stream water concentrations of phosphate, suspended solids and dissolved oxygen. And finally, the "ecological integrity" was represented by the he North Invertebrate Portuguese Index. The results unequivocally showed that point source emissions in the Sabor (except industrial N) and stream water contamination in the Ave determine biodiversity loss. These contrasting influences suggest that Ave basin has evolved from a catchment where man once produced localized negative effects on stream ecological integrity (a condition still observed in the Sabor basin) to a catchment where the dense human occupation has covered the entire area with urban contaminant sources, somewhat generalizing the local effects. The attribution of local effects to biodiversity loss in the rural catchment and of regional effects in the urban catchment is confirmed by the results of a study covering the entire planet.

Reproductive potential and performance of fertility preservation strategies in BRCA-mutated breast cancer patients.

Background: Preclinical evidence suggests a possible negative impact of deleterious BRCA mutations on female fertility. However, limited and rather conflicting clinical data are available. This study assessed the reproductive potential and performance of fertility preservation strategies in BRCA-mutated breast cancer patients. Patients and methods: This was a retrospective analysis of two prospective studies investigating oocyte cryopreservation and ovarian tissue cryopreservation in newly diagnosed early breast cancer patients. In the current analysis, baseline anti-Mullerian hormone (AMH) and performance of cryopreservation strategies were compared between patients with or without germline deleterious BRCA mutations. Results: Out of 156 patients included, 101 had known BRCA status of whom 29 (18.6%) were BRCA-mutated and 72 (46.1%) had no mutation. Median age in the entire cohort was 31 years [interquartile range (IQR) 28-33). Median AMH levels were 1.8 µg/l (IQR 1.0-2.7) and 2.6 µg/l (IQR 1.5-4.1) in the BRCA-positive and BRCA-negative cohorts, respectively (P = 0.109). Among patients who underwent oocyte cryopreservation (N = 29), women in the BRCA-positive cohort tended to retrieve (6.5 versus 9; P = 0.145) and to cryopreserve (3.5 versus 6; P = 0.121) less oocytes than those in the BRCA-negative cohort. Poor response rate (i.e. retrieval of ≤4 oocytes) was 40.0% and 11.1% in the BRCA-positive and BRCA-negative cohorts, respectively (P = 0.147). Among patients who underwent ovarian tissue cryopreservation (N = 72), women in the BRCA-positive cohort tended to have a numerically lower number of oocytes per fragment (0.08 versus 0.14; P = 0.193) and per square millimeter (0.33 versus 0.78; P = 0.153) than those in the BRCA-negative cohort. Two BRCA-mutated patients were transplanted after chemotherapy and one delivered at term a healthy baby. No difference between BRCA1- and BRCA2-mutated patients was observed in any of the above-mentioned outcomes. Conclusion: A consistent trend for reduced reproductive potential and performance of cryopreservation strategies was observed in BRCA-mutated breast cancer patients. Independent validation of these results is needed.

Serum YB-1 (Y-box binding protein 1) as a biomarker of bone disease progression in patients with breast cancer and bone metastases.

YB-1 (Y-box binding protein 1) is a multifunctional cold-shock protein that has been implicated in all hallmarks of cancer. Elevated YB-1 protein level was associated with poor prognosis in several types of cancers, including breast cancer (BC), where it is a marker of decreased overall survival (OS) and distant metastasis-free survival across all subtypes. YB-1 is also secreted by different cell types and may act as an extracellular mitogen; however the pathological implications of the secreted form of YB-1 (sYB-1) are unknown. Our purpose was to retrospectively evaluate the association between YB-1 measured by ELISA in serum and disease characteristics and outcomes in patients with BC and bone metastases (BM). In our cohort, sYB-1 was detected in the serum of 22 (50%) patients, and was associated with the presence of extra-bone metastases (p=0.044). Positive sYB-1 was also associated with faster bone disease progression (HR 3.1, 95% CI 1.09-8.95, P=0.033), but no significant differences were observed concerning OS, and time to development of skeletal-related events. Moreover, patients with positive sYB-1 also had higher levels of IL-6, a known osteoclastogenic inducer. Therefore, detection of sYB-1 in patients with BC and BM may indicate a higher tumor burden, in bone and extra-bone locations, and is a biomarker of faster bone disease progression.

Assessing anthropogenic impacts on riverine ecosystems using nested partial least squares regression.

The results of three Partial Least Squares (PLS) regression models were used to gain a holistic view on the consequences of natural processes and anthropogenic pressures for water quality degradation and biodiversity decline in a multi-use watershed. The processes were soil erosion and wildfire risk; the pressures comprised land use conflicts, leachates from domestic and industrial waste, arable farming intensity and livestock density. Water quality was characterized for concentrations of nutrients (nitrate, phosphate), oxygen demands (Biochemical Oxygen Demand - BOD5, Chemical Oxygen Demand - COD) and various metals (e.g., As, Cr). Ecological integrity was assessed by the recently developed MELI (Multiple Ecological Level Index). In total, 18 variables were processed in the regression models. Two models were called "nested models" because they dealt with initial (pressures), intermediate (water quality) and final (MELI) environmental descriptors, used as dependent (MELI, quality) or independent (quality, pressures) variables. The third was called "bypass model" because it dealt solely with initial and final descriptors. Overall, the results of PLS regression linked the ineffective treatment of domestic sewage to water quality and ecological integrity declines in the studied watershed. Put another way, all models recurrently affirmed the major role of local factors, meaning of point source pollution, in determining the quality of stream water and the integrity of freshwater ecosystems. Sources of diffuse pollution were accounted for as contributing factors in the PLS regressions, but their influence was scarcely perceptible in the results. The poor treatment of domestic effluents is a public concern. In their strategic plans for mitigating this problem in the forthcoming years, administrative authorities are concentrated on management initiatives to improve the quality of provided services, instead of considering the construction of new wastewater treatment plants.

Characterization of allele-specific expression of the X-linked gene MAO-A in trophectoderm cells of bovine embryos produced by somatic cell nuclear transfer.

Somatic cell nuclear transfer (SCNT) may affect epigenetic mechanisms and alter the expression of genes related to embryo development and X chromosome inactivation (XCI). We characterized allele-specific expression of the X-linked gene monoamine oxidase type A (MAO-A) in the trophectoderm (TF) of embryos produced by SCNT. Total RNA was isolated from individual biopsies (N = 25), and the allele-specific expression assessed by reverse transcription-polymerase chain reaction-restriction fragment length polymorphism. Both paternal and maternal alleles were expressed in the trophectoderm. However, a higher frequency of the mono-allelic expression of a specific allele was observed (N = 17; 68%), with the remaining samples showing the presence of mRNA from both alleles (N = 8; 32%). Considering that MAO-A is subject to XCI in bovine, our results suggest that SCNT may influence XCI because neither an imprinted (mono-allelic expression in all samples) nor a random (presence of mRNA from both alleles in all samples) pattern of XCI was observed in TF. Due to the importance of XCI in mammalian embryo development and its sensitivity to in vitro conditions, X-linked genes subject to XCI are candidates for use in the development of embryo quality molecular markers for assisted reproduction.

Comparação entre as abordagens lateral direita e mediana ventral em cadelas submetidas à ovariossalpingo-histerectomia / Comparison between the right lateral and ventral median approach in a bitch undergoing ovariosalpinghysterectomy

A fim de comparar as abordagens abdominais, pela linha mediana ventral e lateral direita em cadelas pré-púberes e adultas submetidas a ovariossalpingo-histerectomia, utilizaram-se 28 cadelas hígidas, distribuídas em dois grupos experimentais de igual número: grupo abordagem mediana ventral (AMV) e grupo abordagem lateral direita (ALD), com sete animais adultos e sete animais pré-púberes em cada grupo. O procedimento cirúrgico foi dividido em nove manobras cirúrgicas distintas, e o tempo para conclusão de cada uma delas, suas facilidades e dificuldades, assim como o tempo cirúrgico total, foram determinados. O tempo médio desde o início da incisão da pele até a entrada na cavidade peritoneal foi menor nas cadelas adultas (P≤0,001) e pré-púberes (P≤0,001) do grupo AMV, mas o tempo médio para identificação uterina foi menor nas cadelas pré-púberes (P≤0,001) do grupo ALD. O tempo cirúrgico total foi menor utilizando-se a abordagem lateral direita (grupo ALD) nas cadelas adultas (P≤0,001) e pré-púberes (P≤0,001). Seu uso não se relacionou com complicações cirúrgicas e facilitou a identificação uterina, possibilitando redução no tempo cirúrgico total. Assim, a abordagem lateral direita demonstrou ser uma alternativa segura em cadelas adultas e pré-púberes submetidas à OSH eletiva.

In order to compare the abdominal approaches, through the ventral midline and right lateral in pre-pubertal adult female dogs undergoing ovariohysterectory we used 28 otherwise healthy dogs, divided into two experimental groups of equal number: Ventral Median Approach Group (VMA) and Right Side Approach Group (RSA), with seven adult animals and seven pre-pubertal animals in each group. The surgical procedure was divided into nine different surgical maneuvers, and the time required for completion of each of them, their strengths and difficulties, as well as the total surgical time were determined. The time from the start of the skin incision to the entrance into the peritoneal cavity was lower in adult female dogs (P≤0.001) and pre-pubertal (P≤0.001) in the VAM group, but the time for uterine identification was lower in pre-pubertal female dogs (P≤ 0.001) in the RSA group. The total surgical time was shorter using the right lateral approach (RSA group) in adult (P≤ 0.001) and pre-pubertal (P≤0.001) female dogs. Thus, the right-side approach has proved to be a safe alternative in adult and pre-pubertal dogs undergoing elective OSH. Its use was not associated with surgical complications, and facilitated uterine identification, allowing a reduction in the total surgical time.

O uso do peritônio de paca conservado em solução supersaturada de açúcar a 300 por cento ou glicerina a 98 por cento implantados na parede abdominal de ratos / The use of paca peritoneum preserved in supersaturated sugar solution 300 percent or glycerine 98 percent implanted in the abdominal wall of rats

Na busca de material biológico alternativo para a realização de implantes, objetivou-se com o presente estudo avaliar comparativamente a implantação do peritônio de paca, uma nova opção de biomaterial, conservado em solução supersaturada de açúcar a 300 por cento e conservado em glicerina a 98 por cento na parede abdominal de ratos Wistar. Foram utilizados 60 ratos, machos, da linhagem Wistar, pesando entre 150 e 200 gramas, organizados nos seguintes grupos experimentais: grupo controle (GI), grupo peritônio conservado em solução supersaturada de açúcar a 300 por cento (GII) e grupo peritônio conservado em glicerina a 98 por cento (GIII), cada um com 20 animais. Os grupos GII e GIII receberam o enxerto de peritônio da paca conservado em solução de açúcar 300 por cento e glicerina 98 por cento, respectivamente, e o grupo GI não recebeu a membrana. Cinco ratos de cada grupo foram submetidos à eutanásia em quatro momentos distintos: sete, 15, 30 e 60 dias de pós-operatório para avaliações macroscópicas e microscópicas da interface implante-tecido nativo. Apesar de reações adversas observadas em 57,5 por cento dos animais do grupo GII e GIII, em 95 por cento dos animais desses grupos houve boa cicatrização da membrana. Na análise histológica, verificou-se a presença de grande infiltrado inflamatório nos períodos iniciais (sete e 15 dias) e grande presença de tecido conjuntivo nos momentos finais (30 e 60 dias). Concluiu-se que o peritônio da paca como membrana biológica conservado nos meios estudados pode ser utilizado com segurança na parede abdominal de ratos; ainda, que sua conservação em solução supersaturada de açúcar a 300 por cento permitiu melhor maleabilidade no ato cirúrgico...

In the search for alternative biological material to perform implants, this study aimed to compare the implantation of paca peritoneum, a new biomaterial option, preserved in supersaturated sugar solution 300 percent and preserved in glycerin 98 percent in the abdominal wall of Wistar rats. A total of 60 male rats from the Wistar strain weighing between 150 and 200 grams were housed into three different experimental groups: control group (GI), peritoneum preserved in supersaturated sugar solution 300 percent group (GII) and peritoneum preserved in glycerin 98 percent group (GIII), with 20 animals each. The GII and GIII received the paca peritoneum graft preserved in sugar solution 300 percent and glycerin 98 percent, respectively and group GI did not receive any membrane. Five rats from each group were euthanized at four different times: seven, 15, 30 and 60 days post-surgery for macroscopic and microscopic evaluations in graft-native tissue interface. Despite the adverse reactions observed in 57.5 percent of GII and GIII, there was good healing of the membrane in 95 percent of the animals in these groups. Upon histological examination there was a large presence of inflammatory infiltrates in the initial periods (seven and 15 days) and a large presence of connective tissue in the final stages (30 and 60). It was concluded that the paca peritoneum as biological membrane preserved as presented in this study can be used safely in the abdominal wall of rats, the preservation in supersaturated sugar solution 300 percent allowed better flexibility during surgery...

Vitrification of bovine oocytes at different meiotic stages using the Cryotop method: assessment of morphological, molecular and functional patterns.

This study aimed to investigate the functional, morphological and molecular patterns of bovine oocytes vitrified at different times during in vitro maturation (IVM). Four groups of oocytes were used: non-vitrified control oocytes (CG), oocytes vitrified at 0 h (V0), oocytes vitrified after 8 h of IVM (V8) and oocytes vitrified after 22 h of IVM (V22). After vitrification, the oocytes were warmed and then returned to the incubator to complete a total of 24h of IVM. To evaluate the effect of vitrification, the nuclear maturation and fertilization rates were assessed by lacmoid staining and ultrastructural electron microscopy. The cleavage and blastocyst rates were evaluated at D2, D7 and D8. The expression levels of CASP3, TP53, HDAC2, SUV39H1 and DNMT1 were investigated by RT-qPCR. The nuclear maturation, oocyte fertilization, cleavage and blastocyst rates were higher (P < 0.05) in the CG group (80%; 81.3%; 88.5%; and 35.8%) than in the V0 (44%; 44.6%; 22.7%; and 2.6%), V8 (50%; 63%; 21.5%; and 2.2%) and V22 (55.5%; 66.9%; 24.1%; and 4.6%) groups. Ultrastructural analysis revealed significant damage within the cytoplasm of all vitrified groups, but more severe degeneration was observed in the V22 group. The gene expression profiles were not affected by vitrification (P > 0.05). In conclusion, cytoplasm degeneration seems to be the most severe form of damage caused by vitrification. The use of the Cryotop method for vitrification severely reduces bovine oocyte viability regardless of whether it is performed at GV, GVBD or MII stage.

Avaliação macroscópica e microscópica de implante de Biomembrana em estômago de coelhos (Oryctolagus cuniculus) / Macroscopic and microscopic evaluation biomembrane implant in the stomach of rabbits (Oryctolagus cuniculus)

Neste estudo foi implantado um retalho de biomembrana de látex natural em substituição a um fragmento da parede de estômago de coelhos albinos, raça Nova Zelândia, adultos, machos não castrados (n=12), com o intuito de avaliar o processo de reparação tecidual no que se refere à biocompatibilidade, à capacidade de reparação tecidual e a possíveis complicações. Aos 15, 30 e 60 dias de pós-operatório, os animais foram sacrificados, mediante o emprego de tiopental sódico (200mg kg-1), para posterior avaliação macroscópica e análise histopatológica da interface do implante com o tecido nativo. Macroscopicamente, aos 15, 30 e 60 dias de pós-operatório observou-se presença de aderências na face serosa. Aos 60 dias de pós-operatório, em todos os animais avaliados, a biomembrana não foi encontrada. Sob microscopia de luz, aos 15 e 30 dias, verificaram-se descontinuidade das camadas muscular e mucosa, presença de infiltrado inflamatório polimorfonuclear. Foram visualizados vasos sanguíneos e fibras musculares. Aos 60 dias, as camadas mucosa, muscular estavam completamente reconstituídas. O implante foi biocompatível e forneceu arcabouço para orientação e desenvolvimento das camadas teciduais do estômago, mediante processos de reparação, restabelecendo a estrutura do órgão.

In this study, biomembrane of natural latex was utilized to replace a section of the stomach wall of New Zealand rabbits, adult and non-castrated males (n=12), in order to evaluate the tissue repair process in regards to its biocompatility, scar formation ability and possible complications. The animals were euthanized at fifteen, 30 and 60 days post operation, by use of sodium thiopental (200mg kg-1), followed by macroscopic and histopathological analysis of the implant interface with the native tissue. Macroscopically, at fifteen, 30, and 60 days post operation adherence was observed in the serosal wall. At 60 days post operation, the biomembrane is not in the stomach. Under light microscopy, at fifteen and 30 days, discontinuity of muscle layer and mucosa layer, and presence of polimorfonuclear population of inflammatory cells was observed. New vessels and muscle fibers were observed. At 60 days, the mucosa and muscle layers were complete reconstituted. The implants were biocompatible and had provided the mainframe for orientation and development of the tissue layers through repairing processes, thus reestablishing the organ structure.

Fast development of Pichia pastoris GS115 Mut(+) cultures employing batch-to-batch control and hybrid semi-parametric modeling.

In this paper, we implemented a model-based optimization platform for fast development of Pichia pastoris cultures employing batch-to-batch control and hybrid semi-parametric modeling. We illustrate the methodology with a P. pastoris GS115 strain expressing a single-chain antibody fragment (scFv) by determining the optimal time profiles of temperature, pH, glycerol feeding and methanol feeding that maximize the endpoint scFv titer. The first hybrid model was identified from data of six exploratory experiments carried out in a pilot 50-L reactor. This model was subsequently used to maximize the final scFv titer of the proceeding batch employing a dynamic optimization program. Thereupon, the optimized time profiles of control variables were implemented in the pilot reactor and the resulting new data set was used to re-identify the hybrid model and to re-optimize the next batch. The iterative batch-to-batch optimization was stopped after 4 complete optimized batches with the final scFv titer stabilizing at 49.5 mg/L. In relation to the baseline batch (executed according to the Pichia fermentation guidelines by Invitrogen) a more than fourfold increase in scFv titer was achieved. The biomass concentration at induction and the methanol feeding rate profile were found to be the most critical control degrees of freedom to maximize scFv titer.