Cryopreservation of ovarian tissue followed by transplantation represents a strategy to restore ovarian function and fertility. Stress from cryopreservation-thawing processes can lead to alterations ...and/or damage to mitochondrial structure and functionality. High resolution respirometry and histological analysis were used to evaluate the effect of cryopreservation and transplantation on ovarian tissue. Four different conditions were performed: Fresh non-transplanted tissue, Fresh transplanted tissue, Cryopreserved non-transplanted tissue and Cryopreserved transplanted tissue. All groups were able to respond to the substrates-uncoupler-inhibitor protocol. We found a dramatic decrease in general oxygen consumption in hemi-ovaries submitted to cryopreservation and/or transplantation. The effect of cryopreservation on mitochondrial metabolism was less intense than effect of transplantation, since the transplantation affected all of the mitochondrial states. A total of 2644 follicles were analyzed. Of these, 2198 were classified as morphologically normal. The percentage of morphologically normal follicles was significantly lower in the Cryopreserved transplanted group when compared to the Cryopreserved non-transplanted group and the Fresh transplanted group (p-value < 0.05). Despite decreased follicular viability and mitochondrial activity, the cryopreservation followed by transplantation of ovarian tissue proved feasible for attempts to restore ovarian function.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Is the optimal timing for administering erythropoietin to minimize ischaemic injury in ovarian tissue transplantation before ovary removal for cryopreservation and subsequent transplantation or after ...transplantation?
Thirty Swiss mice (nu/nu) were divided into three groups: treatment control group (n = 10); erythropoietin before harvesting group (EPO-BH) (n = 10) and erythropoietin after transplantation group (EPO-AT) (n = 10). Animals underwent bilateral ovariohysterectomy and their hemiovaries were cryopreserved by slow freezing. At the same time, previously cryopreserved hemiovaries were transplanted subcutaneously in the dorsal region. Erythropoietin (250 IU/kg) and sterile 0.9% saline solution were administered every 12/12 h over 5 consecutive days in the EPO-AT and EPO-BH groups, respectively.
Administration of erythropoietin in the EPO-AT group improved the viability of ovarian follicles, reducing degeneration and increasing the number of morphologically normal growing follicles at 14 days after transplantation compared with the EPO-BH group (P = 0.002). This group also showed higher percentages of proliferative follicles at 7 days after transplantation (P ≤ 0.03), increased blood vessel count (P ≤ 0.03) and greater tissue area occupied by blood vessels at days 7 and 14 after transplantation (P ≤ 0.03), compared with hormone administration before cryopreservation (EPO-BH group) and the treatment control group. Additionally, treatment with erythropoietin before or after transplantation reduced fibrotic areas at 7 days after transplantation (P = 0.004).
Erythropoietin treatment after transplantation reduced ischaemic damage in transplanted ovarian tissue, increased angiogenesis, maintenance of ovarian follicle proliferation and reduced fibrosis areas in the grafted tissue.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Many feline species are currently threatened with extinction. Therefore, germplasm bank establishment has become imperative. However, cryoinjury and ischemia-reperfusion injury pose significant ...obstacles to both cryopreservation and xenotransplantation. In this regard, erythropoietin (Epo) represents a potential alternative strategy due to its properties. This study aimed to assess the incubation of domestic cat ovarian tissue in Epo, both before and after cryopreservation, and investigate its effectiveness in promoting revascularization following xenotransplantation. Sixteen ovaries from 8 healthy cats were sliced following elective bilateral ovariohysterectomy (OHE). Subsequently, 8 fragments measuring 3 mm³ each were obtained from the cortical region of each ovary. The fragments were allocated into 3 treatment groups: Cryo group, fragments were cryopreserved, thawed and immediately transplanted; Cryo + Epo group, fragments were first cryopreserved in nitrogen, thawed, incubated in Epo (100 IU) for 2h and transplanted; and the Epo + Cryo group, in which fragments were first incubated in Epo (100 IU) for 2h, cryopreserved, thawed and immediately transplanted. The fragments were then xenotransplanted into the dorsal subcutaneous region of ovariectomized female nude mice and retrieved at 7, 14, 21, and 28 days post-transplantation. The results indicated that Epo effectively enhanced follicular survival, preservation of viability, and tissue revascularization. The Epo + Cryo group displayed better revascularization rates on D14 and D21 post-transplantation and an increase in primordial and growing follicles on D28, the Cryo + Epo group exhibited significantly more follicles on D14 and D21, with fewer degenerated follicles.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Human reproduction presents a challenge for our species, as evidenced by the escalating rates of infertility. This trend has prompted inquiries into diverse strategies aimed at mitigating infertility ...and enhancing conception rates. Despite the extensive research on advanced maternal age as a risk factor for reproductive outcomes, paternal age has historically garnered comparatively less attention. The aim of this study was to assess the impact of paternal age on embryos and its subsequent repercussions on fertilization rate, biochemical pregnancy, clinical pregnancy, and live birth rate in individuals undergoing assisted reproductive treatment in a public reproductive center located in Brazil.OBJECTIVEHuman reproduction presents a challenge for our species, as evidenced by the escalating rates of infertility. This trend has prompted inquiries into diverse strategies aimed at mitigating infertility and enhancing conception rates. Despite the extensive research on advanced maternal age as a risk factor for reproductive outcomes, paternal age has historically garnered comparatively less attention. The aim of this study was to assess the impact of paternal age on embryos and its subsequent repercussions on fertilization rate, biochemical pregnancy, clinical pregnancy, and live birth rate in individuals undergoing assisted reproductive treatment in a public reproductive center located in Brazil.This investigation adopted a retrospective cohort, cross-sectional, analytical design, utilizing the analysis of secondary data, covering the period from July 2015 to July 2021.METHODSThis investigation adopted a retrospective cohort, cross-sectional, analytical design, utilizing the analysis of secondary data, covering the period from July 2015 to July 2021.A total of 350 couples grappling with infertility and undergoing intrauterine insemination (IUI), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI) were included in the analysis. Examination of age groups revealed a notable correlation between the ages of women and men (correlation coefficient R=0.12, p<0.0001). In the analysis of IVF techniques, a discernible trend towards a negative correlation with paternal age was observed, signifying that higher paternal age was linked to lower fertilization rates (p=0.004).RESULTSA total of 350 couples grappling with infertility and undergoing intrauterine insemination (IUI), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI) were included in the analysis. Examination of age groups revealed a notable correlation between the ages of women and men (correlation coefficient R=0.12, p<0.0001). In the analysis of IVF techniques, a discernible trend towards a negative correlation with paternal age was observed, signifying that higher paternal age was linked to lower fertilization rates (p=0.004).Advanced paternal age significantly impacts full-term birth rates in IVF procedures, emphasizing the need for preconception public health advisories that underscore the risks associated with delaying parenthood for both men and women, particularly among those necessitating assisted reproductive techniques.CONCLUSIONSAdvanced paternal age significantly impacts full-term birth rates in IVF procedures, emphasizing the need for preconception public health advisories that underscore the risks associated with delaying parenthood for both men and women, particularly among those necessitating assisted reproductive techniques.
The dynamics underlying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reinfection remain poorly understood. We identified a small cluster of patients in Brazil who experienced 2 ...episodes of coronavirus disease (COVID-19) in March and late May 2020. In the first episode, patients manifested an enhanced innate response compared with healthy persons, but neutralizing humoral immunity was not fully achieved. The second episode was associated with different SARS-CoV-2 strains, higher viral loads, and clinical symptoms. Our finding that persons with mild COVID-19 may have controlled SARS-CoV-2 replication without developing detectable humoral immunity suggests that reinfection is more frequent than supposed, but this hypothesis is not well documented.
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DOBA, IZUM, KILJ, NUK, ODKLJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
► We isolated of the S. cerevisiae LBM-1 strain, which is capable of growth at 42°C. ► We compared the capacity of different yeast strains to ferment sugar cane bagasse. ► Presaccharification stage ...is key to increasing ethanol yields. ► Use of thermotolerant yeasts increase the yield of the SSF process.
Ethanol can be produced from cellulosic biomass in a process known as simultaneous saccharification and fermentation (SSF). The presence of yeast together with the cellulolytic enzyme complex reduces the accumulation of sugars within the reactor, increasing the ethanol yield and saccharification rate. This paper reports the isolation of Saccharomyces cerevisiae LBM-1, a strain capable of growth at 42°C. In addition, S. cerevisiae LBM-1 and Kluyveromyces marxianus UFV-3 were able to ferment sugar cane bagasse in SSF processes at 37 and 42°C. Higher ethanol yields were observed when fermentation was initiated after presaccharification at 50°C than at 37 or 42°C. Furthermore, the volumetric productivity of fermentation increased with presaccharification time, from 0.43g/L/h at 0h to 1.79g/L/h after 72h of presaccharification. The results suggest that the use of thermotolerant yeasts and a presaccharification stage are key to increasing yields in this process.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Lignocellulosic wastes can be potentially converted into several bioproducts such as glucose, xylo‐oligosaccharides, and bioethanol. Certain processes, such as enzymatic hydrolysis, are generally ...needed to convert biomass into bioproducts. The present study investigated the production of xylanases and cellulases by Streptomyces thermocerradoensis I3 under solid‐state fermentation (SSF), using wheat bran as a low‐cost medium. The activities of xylanase and carboxymethyl cellulase (CMCase) were evaluated until 96 hr of incubation. The highest enzyme activity was observed after 72 hr of incubation. The crude enzyme extract was sequentially filtered, first using a 50 kDa filter, followed by a 30 kDa filter. Fraction 3 (F3) exhibited activities of both xylanase and CMCase. Xylanase and CMCase showed optimum activity at 70°C and pH 6.0 and 55°C and pH 6.0, respectively. The zymogram analysis showed a single activity band with a molecular mass of approximately 17 kDa. These findings provide strong evidence that the enzyme is a bifunctional xylanase/endoglucanase. This enzyme improved the saccharification of sugarcane bagasse by 1.76 times that of commercial cellulase. This enzyme has potential applications in various biotechnological procedures.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is already responsible for far more deaths than previous pathogenic coronaviruses (CoVs) from 2002 and 2012. The identification of ...clinically approved drugs to be repurposed to combat 2019 CoV disease (COVID-19) would allow the rapid implementation of potentially life-saving procedures. The major protease (Mpro) of SARS-CoV-2 is considered a promising target, based on previous results from related CoVs with lopinavir (LPV), an HIV protease inhibitor. However, limited evidence exists for other clinically approved antiretroviral protease inhibitors. Extensive use of atazanavir (ATV) as antiretroviral and previous evidence suggesting its bioavailability within the respiratory tract prompted us to study this molecule against SARS-CoV-2. Our results show that ATV docks in the active site of SARS-CoV-2 Mpro with greater strength than LPV, blocking Mpro activity. We confirmed that ATV inhibits SARS-CoV-2 replication, alone or in combination with ritonavir (RTV) in Vero cells and a human pulmonary epithelial cell line. ATV/RTV also impaired virus-induced enhancement of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) levels. Together, our data strongly suggest that ATV and ATV/RTV should be considered among the candidate repurposed drugs undergoing clinical trials in the fight against COVID-19.
Abstract
Background
Current approaches of drug repurposing against COVID-19 have not proven overwhelmingly successful and the SARS-CoV-2 pandemic continues to cause major global mortality. SARS-CoV-2 ...nsp12, its RNA polymerase, shares homology in the nucleotide uptake channel with the HCV orthologue enzyme NS5B. Besides, HCV enzyme NS5A has pleiotropic activities, such as RNA binding, that are shared with various SARS-CoV-2 proteins. Thus, anti-HCV NS5B and NS5A inhibitors, like sofosbuvir and daclatasvir, respectively, could be endowed with anti-SARS-CoV-2 activity.
Methods
SARS-CoV-2-infected Vero cells, HuH-7 cells, Calu-3 cells, neural stem cells and monocytes were used to investigate the effects of daclatasvir and sofosbuvir. In silico and cell-free based assays were performed with SARS-CoV-2 RNA and nsp12 to better comprehend the mechanism of inhibition of the investigated compounds. A physiologically based pharmacokinetic model was generated to estimate daclatasvir’s dose and schedule to maximize the probability of success for COVID-19.
Results
Daclatasvir inhibited SARS-CoV-2 replication in Vero, HuH-7 and Calu-3 cells, with potencies of 0.8, 0.6 and 1.1 μM, respectively. Although less potent than daclatasvir, sofosbuvir alone and combined with daclatasvir inhibited replication in Calu-3 cells. Sofosbuvir and daclatasvir prevented virus-induced neuronal apoptosis and release of cytokine storm-related inflammatory mediators, respectively. Sofosbuvir inhibited RNA synthesis by chain termination and daclatasvir targeted the folding of secondary RNA structures in the SARS-CoV-2 genome. Concentrations required for partial daclatasvir in vitro activity are achieved in plasma at Cmax after administration of the approved dose to humans.
Conclusions
Daclatasvir, alone or in combination with sofosbuvir, at higher doses than used against HCV, may be further fostered as an anti-COVID-19 therapy.
(domestic dog) represents a reliable sentinel for the occurrence of a well-established transmission cycle of
among wild mammals in the surroundings and, consequently, where the risk of human ...infection exists. Serological diagnosis is the chosen method to identify
infection in dogs that, in Brazil, rarely present positive parasitological tests. The use of recombinant chimeric parasitic antigens results in a sensitive and specific serological diagnostic test in contrast to the use of crude
antigens. Our objective was to evaluate the Chagas/Bio-Manguinhos Lateral Flow Immunochromatographic Rapid Test (Chagas-LFRT) for the diagnosis of
infection in domestic dogs and the potential of application of this diagnostic platform to wild canid species. Two recombinant proteins (IBMP-8.1 and IBMP-8.4) that displayed the best performance in the enzyme immunoassay (ELISA) in previous studies were tested in a platform with two diagnostic bands. A panel of 281 dog serum samples was evaluated: 133 positive for
by serological diagnosis, including 20 samples with positive blood cultures belonging to different discrete typing units (DTUs); 129 negative samples; and 19 samples from dogs infected by other trypanosomatids:
,
,
and
, in addition to samples infected by
,
and
sp. that were employed to evaluate eventual cross-reactions. We also evaluated the Chagas-LFRT to detect
infection in 9 serum samples from six wild canid species. We observed that the intensity pattern of the bands was directly proportional to the serological titer observed in IFAT. The sensitivity was 94%, the specificity was 91% according to the ROC curve, and the defined cutoff was an optical density of 4.8. The agreement obtained was considered substantial by the kappa analysis (84%). From
positive hemoculture samples, 88.9% were positive by Chagas-LFRT. The test was efficient in recognizing infections by five of the six
DTUs. Cross-reactions were not observed in infections by
,
,
and
; however, they were observed in sera of dogs infected by
,
sp. and
sp. A strong reaction was observed when serum samples from wild canids were submitted to the Protein A affinity test, confirming its applicability for these species. This test will allow rapid preventive actions in areas with high risk to the emergence of Chagas disease in a safer, reliable, low-cost and immediate manner, without the need for more complex laboratory tests.
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