Diversity and probiotic characterisation of yeast isolates in the bovine gastrointestinal tract.

The use of yeasts as a feed supplement for cattle can promote animal development and performance. However, for the positive results to be consistent, strains with probiotic properties must be selected. The objective of this study was to isolate and identify yeasts present in the bovine feces and evaluate their probiotic potential together with strains previously isolated from the rumen (preliminary study). A total of 193 isolates were studied, including 139 isolates (19 species) from fecal samples from 11 different animals (Bos taurus and Bos indicus) and 54 strains previously isolated from rumen fluid (Bos taurus). The yeast population in the feces ranged from 3.51 to 4.99 log CFU/g, with Candida pararugosa being the most abundant (isolated from the feces of six samples analysed). Isolates were selected that had negative results in the safety tests (hemolytic activity, DNAse, and gelatinase) and had percentages greater than 35 and 70% for hydrophobicity and auto-aggregation, respectively. In addition, selected isolates had percentages greater than 77.7 and 74.7% for coaggregation with pathogenic strains of Escherichia coli and Clostridium perfringens, respectively. The isolates with percentage growth at 39 °C greater than 64.6% and viability greater than 96.7% were selected for survival testing under bovine gastrointestinal conditions. After the tests, the seven best isolates were selected, belonging to the species Candida pararugosa (L60, CCMA 928 and CCMA 930) and Pichia kudriavzevii (L97, L100, CCMA904, CCMA 907). The selected isolates were exopolysaccharide producers. Based on the results of the evaluated properties, the seven selected isolates were classified as potential probiotics for cattle.

Criteria for lactic acid bacteria screening to enhance silage quality.

The main challenge of ensiling is conserving the feed through a fermentative process that results in high nutritional and microbiological quality while minimizing fermentative losses. This challenge is of growing interest to farmers, industry and research and involves the use of additives to improve the fermentation process and preserve the ensiled material. Most studies involved microbial additives; lactic acid bacteria (LAB) have been the focus of much research and have been widely used. Currently, LABs are used in modern and sustainable agriculture because of their considerable potential for enhancing human and animal health. Although the number of studies evaluating LABs in silages has increased, the potential use of these micro-organisms in association with silage has not been adequately studied. Fermentation processes using the same strain produce very different results depending on the unique characteristics of the substrate, so the choice of silage inoculant for different starting substrates is of extreme importance to maximize the nutritional quality of the final product. This review describes the current scenario of the bioprospecting and selection process for choosing the best LAB strain as an inoculant for ensiling. In addition, we analyse developments in the fermentation process and strategies and methods that will assist future studies on the selection of new strains of LAB as a starter culture or inoculant.

Violacein antimicrobial activity on Staphylococcus epidermidis and synergistic effect on commercially available antibiotics.

AIMS: The study aimed to assess whether violacein has antimicrobial activity on Staphylococcus epidermidis and synergistically modulates the action of commercially available antimicrobial drugs. METHODS AND RESULTS: Violacein showed excellent antimicrobial activity on biofilm-forming and nonbiofilm-forming S. epidermidis strains (ATCC 35984) (ATCC 12228), with bacteriostatic (MIC = 20 µg ml-1 and 10 µg ml-1 respectively) and bactericidal effects (MBC = 20 µg ml-1 for both strains), observed in short periods of exposure. The violacein bactericidal concentration led to S. epidermidis death after 2-3 h of exposure. Additionally, violacein synergistically modulated the activity of different antimicrobial classes on S. epidermidis ATCC 12228 (81·8%; n = 9) and on S. epidermidis ATCC 35984 (54·5%; n = 6), reducing the MIC of these antibiotics by up to 16-fold. CONCLUSION: Violacein shows excellent antimicrobial activity on S. epidermidis strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Violacein shows the potential for the development of a new drug for the treatment of infections caused by S. epidermidis.

A prognostic regulatory pathway in stage I epithelial ovarian cancer: new hints for the poor prognosis assessment.

BACKGROUND: Clinical and pathological parameters of patients with epithelial ovarian cancer (EOC) do not thoroughly predict patients' outcome. Despite the good outcome of stage I EOC compared with that of stages III and IV, the risk assessment and treatments are almost the same. However, only 20% of stage I EOC cases relapse and die, meaning that only a proportion of patients need intensive treatment and closer follow-up. Thus, the identification of cell mechanisms that could improve outcome prediction and rationalize therapeutic options is an urgent need in the clinical practice. PATIENTS AND METHODS: We have gathered together 203 patients with stage I EOC diagnosis, from whom snap-frozen tumor biopsies were available at the time of primary surgery before any treatment. Patients, with a median follow-up of 7 years, were stratified into a training set and a validation set. RESULTS AND CONCLUSIONS: Integrated analysis of miRNA and gene expression profiles allowed to identify a prognostic cell pathway, composed of 16 miRNAs and 10 genes, wiring the cell cycle, 'Activins/Inhibins' and 'Hedgehog' signaling pathways. Once validated by an independent technique, all the elements of the circuit resulted associated with overall survival (OS) and progression-free survival (PFS), in both univariate and multivariate models. For each patient, the circuit expressions have been translated into an activation state index (integrated signature classifier, ISC), used to stratify patients into classes of risk. This prediction reaches the 89.7% of sensitivity and 96.6% of specificity for the detection of PFS events. The prognostic value was then confirmed in the external independent validation set in which the PFS events are predicted with 75% sensitivity and 94.7% specificity. Moreover, the ISC shows higher classification performance than conventional clinical classifiers. Thus, the identified circuit enhances the understanding of the molecular mechanisms lagging behind stage I EOC and the ISC improves our capabilities to assess, at the time of diagnosis, the patient risk of relapse.

Low dose rate γ-irradiation protects fruit fly chromosomes from double strand breaks and telomere fusions by reducing the esi-RNA biogenesis factor Loquacious.

It is still continuously debated whether the low-dose/dose-rate (LDR) of ionizing radiation represents a hazard for humans. Model organisms, such as fruit flies, are considered valuable systems to reveal insights into this issue. We found that, in wild-type Drosophila melanogaster larval neuroblasts, the frequency of Chromosome Breaks (CBs), induced by acute γ-irradiation, is considerably reduced when flies are previously exposed to a protracted dose of 0.4 Gy delivered at a dose rate of 2.5 mGy/h. This indicates that this exposure, which is associated with an increased expression of DNA damage response proteins, induces a radioadaptive response (RAR) that protects Drosophila from extensive DNA damage. Interestingly, the same exposure reduces the frequency of telomere fusions (TFs) from Drosophila telomere capping mutants suggesting that the LDR can generally promote a protective response on chromatin sites that are recognized as DNA breaks. Deep RNA sequencing revealed that RAR is associated with a reduced expression of Loquacious D (Loqs-RD) gene that encodes a well-conserved dsRNA binding protein required for esiRNAs biogenesis. Remarkably, loss of Loqs mimics the LDR-mediated chromosome protection as it decreases the IR-induced CBs and TFs frequency. Thus, our molecular characterization of RAR identifies Loqs as a key factor in the cellular response to LDR and in the epigenetic routes involved in radioresistance.

Carbapenem-resistant Klebsiella pneumoniae in ICU-admitted COVID-19 patients: Keep an eye on the ball.

Here we report on seven intensive care unit (ICU) patients with coronavirus disease 2019 (COVID-19)-related acute respiratory distress syndrome (ARDS) who developed positive rectal swabs and invasive infections due to carbapenemase-producing Klebsiella pneumoniae (CP-Kp). Notwithstanding the infection prevention measures introduced during the COVID-19 pandemic and changes in the hospitalised population, attention to CP-Kp infections must remain high, especially in the critically ill setting.

Violacein antimicrobial activity on Staphylococcus epidermidis biofilm.

The aim of this study was to evaluate the antimicrobial potential of violacein (VIO) on Staphylococcus epidermidis biofilm. The minimum biofilm inhibition concentration (MBIC) and minimum biofilm eradication concentration (MBEC) were determined, as well as the effect of VIO exposure time on microbial viability in mature biofilm. Violacein showed good antibiofilm action, inhibiting biofilm formation and eradicating mature biofilm of S. epidermidis at concentrations of 20 µg.mL-1 and 160 µg.mL-1, respectively. At concentrations equal to MBEC and 2x MBEC, the biofilm was eradicated in 3 h and 2h30min of incubation, respectively.When evaluating VIO modulating effect on the action of clinically-used drugs (vancomycin, cefepime, ciprofloxacin and meropenem), especial synergism was observed in the violacein-ciprofloxacin association, it can completely erradicated the mature biofilm at the concentration of 1/2xMBEC and 1/4xMBEC, respectively. VIO shows good antimicrobial action on S. epidermidis biofilm and has the potential to synergistically modulate the activity of clinically-used antimicrobials.

Low-dose Spinal Block With Continuous Epidural Infusion for Renal Transplantation in a Patient With Alport Syndrome: A Case Report.

OBJECTIVE: We aim to describe management of a patient receiving renal transplantation for chronic renal failure due to Alport syndrome with low dose of intrathecal bupivacaine and continuous epidural infusion of local anesthetic. CASE REPORT: A 38-years-old man with chronic renal failure secondary to Alport syndrome underwent kidney transplantation. Because of a high risk of respiratory and cardiovascular complications related to the patient's baseline lung disease and abnormalities in heart conduction, we selected combined spinal-epidural anesthesia. The block was ultrasound-guided and performed at the T12-L1 interspace with 4.5 mg of 0.5% intrathecal hyperbaric bupivacaine followed by a continuous epidural infusion of 0.5% levobupivacaine mixed with 25 µg of Fentanyl at the initial rate of 8 mL/h. Sensory block to T5-T6 was obtained within 10 minutes. The patient then received mild sedation with Propofol and Remifentanil. Methylprednisolone and diuretics were administered before vascular unclamping according to our internal protocol. Surgery lasted 3 hours with no clinical or procedural complication. CONCLUSIONS: Although renal transplantation is usually performed under general anesthesia, in a particularly complex patient with chronic renal failure, chronic obstructive pulmonary disease and a worsened respiratory mechanics, we applied a combined approach with a low dose of intrathecal bupivacaine and continuous epidural infusion of local anesthetic. The technique did not affect hemodynamics while having a positive impact on recovery of function of the transplanted organ with rapid improvement of urine output, serum creatinine, and blood urea nitrogen levels.

A data-driven network model of primary myelofibrosis: transcriptional and post-transcriptional alterations in CD34+ cells.

microRNAs (miRNAs) are relevant in the pathogenesis of primary myelofibrosis (PMF) but our understanding is limited to specific target genes and the overall systemic scenario islacking. By both knowledge-based and ab initio approaches for comparative analysis of CD34+ cells of PMF patients and healthy controls, we identified the deregulated pathways involving miRNAs and genes and new transcriptional and post-transcriptional regulatory circuits in PMF cells. These converge in a unique and integrated cellular process, in which the role of specific miRNAs is to wire, co-regulate and allow a fine crosstalk between the involved processes. The PMF pathway includes Akt signaling, linked to Rho GTPases, CDC42, PLD2, PTEN crosstalk with the hypoxia response and Calcium-linked cellular processes connected to cyclic AMP signaling. Nested on the depicted transcriptional scenario, predicted circuits are reported, opening new hypotheses. Links between miRNAs (miR-106a-5p, miR-20b-5p, miR-20a-5p, miR-17-5p, miR-19b-3p and let-7d-5p) and key transcription factors (MYCN, ATF, CEBPA, REL, IRF and FOXJ2) and their common target genes tantalizingly suggest new path to approach the disease. The study provides a global overview of transcriptional and post-transcriptional deregulations in PMF, and, unifying consolidated and predicted data, could be helpful to identify new combinatorial therapeutic strategy. Interactive PMF network model: http://compgen.bio.unipd.it/pmf-net/.

Surgical results and prognostic factors in early non-small cell lung cancer.

BACKGROUND: We attempted to clarify the prognostic value of tumor size (maximum, 3 cm), the evidence of invasion proximal to a lobar bronchus at least 2 cm distal to the carina, and the absence or presence of visceral pleura invasion in patients with completely resected non-small cell lung carcinoma without lymph node invasion or satellite lesions (T1 N0 M0, T2 N0 M0). METHODS: The study included 158 patients. Four patients were excluded due to postoperative mortality (2.5%). The variables selected for the survival study were sex, age, symptoms presence or absence, bronchial invasion level (evidence or not of invasion proximal to a lobar bronchus at least 2 cm distal to the carina), pulmonary location, pneumonectomy or lesser resection, cell type, squamous or nonsquamous, tumor size, invasion or not of the visceral pleura, and T1 or T2 status. RESULTS: The overall survival rate in this series was 74% at 5 years and 60% at 10 years. Only the tumor size had a significant influence on survival (p = 0.0092). Patients with a tumor less than 2 cm in diameter did better (p = 0.0023). CONCLUSIONS: These observations suggest that it will be necessary to further research in clarifying the prognostic value of the bronchial invasion level and of the degree of the visceral pleura invasion and its implications when classifying a tumor as T1 or T2.

Ultrasonic vocalization in rat pups: effects of early postnatal exposure to haloperidol.

The effects of prolonged postnatal administration of haloperidol (H) on ultrasonic vocalization elicited by the removal of rat pups from their nest were investigated. The results show that the number of ultrasonic calls was significantly reduced by H exposure from the 8th until the 14th day after birth. Conversely, this neuroleptic significantly increased the duration of ultrasound from the 4th up to the 16th day of age. Moreover, changes in the frequency of calls were produced by early postnatal treatment with H. These alterations could be due to an impaired functional maturation of the dopaminergic system produced by neonatal exposure to H. Furthermore, the present data suggest that ultrasonic vocalization may be considered as an early sensitive indicator of subtle changes elicited by the postnatal treatment with a dopamine receptor blocking agent at dose levels below those associated with overt signs of neurotoxicity.

Sound levels in rooms housing laboratory animals: an uncontrolled daily variable.

High sound levels are known to have adverse effects on the behaviour and physiology of laboratory animals, yet their acoustic environment is rarely monitored. In particular, high-frequency sounds that are above the limit of human hearing, but are well within the limits of many laboratory species (i.e., ultrasounds), are usually ignored. In this study, the acoustic environment of laboratory animals was investigated in a variety of different animal facilities. Sound pressure levels (dB SPL) were monitored for periods up to 24 h over two frequency ranges: a relatively low range (0.01-12.5 kHz), and a high range (12.5-70 kHz). While background sound levels in undisturbed situations were generally low (i.e., below 50 dB SPL), marked increases in sound levels often occurred during the working day, producing characteristic daily variations in the sound profile. Peak SPLs commonly reached values of 80-95 dB in the low-frequency range and 50-75 dB in the higher range. In most cases, sound levels were low over weekends. The results suggested that human activities were a very important source of sound in most animal facilities. In a few situations (e.g., rabbits, marmosets, dogs), the animals themselves provided a significant contribution to the acoustic environment. It is clear that the acoustic environment of laboratory animals is a daily variable that is usually uncontrolled and that may have important implications for behavioural and physiological experiments and for animal welfare.

The emission and elicitation of mouse ultrasonic vocalizations: the effects of age, sex and gonadal status.

Subject' mice of varying gonadal status (castrate males; intact or neonatally gonadectomized females and males) were paired for 3-min with intact 'stimulus' females and ultrasonic vocalizations were monitored. Vocalization patterns from home cages differed from the test pairings. The results suggested that the age, experience and gonadal status of the subject influenced the vocalizations from the pair. As the source of ultrasonic calls from these vocally intact pairs could not be individually identified, the 'subjects' were paired with a range of ultrasonically silent (inferior laryngeal nerve-transected) stimulus animals. Vocalizations were detected from all combinations of animals. Gonadally intact females were most effective in eliciting ultrasonic vocalizations from the subjects and gonadally intact males were least effective. The responses of castrate males were lower than from intact males. Anesthetized adults of either sex elicited only poor vocalization responses from other adults. Ultrasonic calls have often previously been studied using vocally intact 'subject' and 'stimulus' animals: the present results confirm the difficulty of establishing who is who in such situations.

Long-term monitoring of mouse ultrasonic vocalizations.

The temporal pattern of ultrasonic vocalizations by mice in an undisturbed 'home' environment can now be assessed using a system based on amplitude discrimination. Within a chosen frequency band, vocalizations of sufficient intensity are detected by an amplitude discriminator. The output from a pulse generator is sent to a microcomputer which records the time of the incoming event. The system has been validated for monitoring ultrasonic vocalizations in the mouse.

Comparison of the oestrogenic effects of infant milk formulae, oestradiol and the phytoestrogen coumestrol delivered continuously in the drinking water to ovariectomised mice.

The potential oestrogenic effects of infant milk formulae, coumestrol and oestradiol delivered in the drinking water were investigated in ovariectomised mice. None of the infant formulae tested (three soya, two cow's milk) produced any uterotrophic or mitotic responses in the reproductive tract, although the soya milks displayed weak oestrogenic activity in vitro. Studies of the interactions between coumestrol and oestradiol were undertaken to investigate claims that phytoestrogens may act as oestrogen antagonists. The responses to coumestrol (100 g/ml drinking water) and 17-oestradiol (100 ng/ml) given separately were similar. Combined administration begun simultaneously produced only additive effects on uterine weight and cell proliferation in the vagina and uterus. While pretreatment with coumestrol for 24 h reduced the mitotic response of the uterus 48 h after placement of an oestradiol implant, the uterine weight increase was unaffected and the apparent reduction in mitoses reflected the natural fluctuations in the underlying cycle of cell proliferation. These studies indicate that coumestrol acts as a typical oestrogen and shows only additive effects with oestradiol. The results also indicate that infant soya milk formulae do not constitute a large enough source of oestrogenic compounds to invoke oestrogenic effects in the reproductive tract of mature mice.

The effect of 22 kHz calls and artificial 38 kHz signals on activity in rats.

Ultrasonic calls at 22 kHz are emitted by adult rats Rattus norvegicus when submissive, after and sometimes before copulation and in aversive situations, but the function of these calls, if any, is not known. This study examined the effect of 22 kHz calls on the behaviour of individuals. Rats were exposed singly for 5 min to a tape-recorded test signal; 22 kHz calls or artificial 38 kHz signals or tape noise. All rats were exposed to tape noise before and after the test signal. Both of the ultrasonic signals, and particularly the 22 kHz calls, decreased locomotor activity in rats during replay and for 5 min afterwards. Rats sniffed the loudspeaker less during the replay of 22 kHz calls than during the other test signals. These results complement others showing that constant frequency ultrasound from visual display units and oscilloscopes also depresses activity. In natural aggressive situations 22 kHz calls could allow a subordinate to escape and prevent the dominant wasting time in pursuit. In the laboratory exposure of rats to natural or artificial ultrasound could affect ongoing or subsequent behaviour and so be a confounding experimental variable.

Behaviour of rats in their home cages: daytime variations and effects of routine husbandry procedures analysed by time sampling techniques.

The behaviour of laboratory rats in their home cages was observed on both the mornings and the afternoons of days when cages were cleaned and compared to days when cages were not cleaned. Two different time sampling methods, 'instantaneous sampling' and 'one/zero sampling', were used and compared. In general the rats were more active in the mornings than in the afternoons. Activity, particularly locomotion and that associated with manipulation of the bedding was increased during both the mornings and the afternoons of cleaning days. Defaecation also increased on cleaning days whereas sitting decreased. The cleaning regime appeared to have a greater effect on behaviour than did time of day and the effect of cleaning lasted for several hours after the procedure had been completed. The implications for experimental design are discussed.

Environmental ultrasound in laboratories and animal houses: a possible cause for concern in the welfare and use of laboratory animals.

Many laboratory animals are known to be sensitive to sounds (ultrasounds) beyond the nominal upper limit (20 kHz) of the human hearing range. Sources of sound in laboratories and animal houses were examined to determine the extent of ambient ultrasound. Of 39 sources monitored, 24 were found to emit ultrasonic sounds. Many of these (e.g. cage washers and hoses) also produced sound in the audible range. Running taps, squeaky chairs and rotating glass stoppers created particularly high sound pressure levels and contained frequencies to over 100 kHz. The oscilloscopes and visual display units investigated provided particular cause for concern as they emitted sounds that were entirely ultrasonic and therefore were apparently silent. Ambient ultrasound therefore appears to be common in laboratories and animal houses. It is suggested that its effect on laboratory animals should be investigated and guidelines on acceptable levels be formulated.

[Individualized prognosis in non-small-cell carcinoma. A multivariate approach]. / Pronóstico individualizado en el carcinoma de pulmón no microcítico. Una aproximación multivariada.

With the aim of providing a system to give individualized initial prognosis for patients with non-small cell lung cancer (NSLC), we performed a multivariate discriminant analysis with combinations of various prognostic factors, studying 93 patients with diagnoses of stage II NSLC and complete followup information. All had undergone surgery at our hospital. Survival longer than or less than one year was defined as the dependent variable: independent variables more clinical, analytical, lung function, histological, anatomical and surgical data. Among the 31.2% of patients who died within one year, tumor size was noticeably greater (6.0 +/- 2.1 cm versus 4.8 +/- 2.0 cm; p = 0.11) and serum albumin was lower (3.7 +/- 0.7 g/dl versus 3.9 +/- 0.6 g/dl; p = 0.039); we found no significant differences among the remaining independent variables in the preliminary univariate analysis. The linear function obtained with discriminant analysis allowed us to classify the patients correctly in 87.1% of cases, with a diagnostic sensitivity of 79.3%, specificity of 90.6%, positive predictive value of 79.3% and negative predictive value of 90.6% for patients who died during the first year. The independent variables that were associated with poor prognosis were large tumor size, long duration of symptoms, low albumin level, high alkaline phosphate level, presence of 2 or more N1 affected nodes, pneumonectomy and presence of perioperative complications. Our findings allow us to conclude that the simultaneous analysis of a variety of prognostic factors can help to give an accurate prognosis for individual patients with the same anatomical stage classification.