External and internal triggers of cell death in yeast

In recent years, yeast was confirmed as a useful eukaryotic model system to decipher the complex mechanisms and networks occurring in higher eukaryotes, particularly in mammalian cells, in physiological as well in pathological conditions. This article focuses attention on the contribution of yeast in the study of a very complex scenario, because of the number and interconnection of pathways, represented by cell death. Yeast, although it is a unicellular organism, possesses the basal machinery of different kinds of cell death occurring in higher eukaryotes, i.e., apoptosis, regulated necrosis and autophagy. Here we report the current knowledge concerning the yeast orthologs of main mammalian cell death regulators and executors, the role of organelles and compartments, and the cellular phenotypes observed in the different forms of cell death in response to external and internal triggers. Thanks to the ease of genetic manipulation of this microorganism, yeast strains expressing human genes that promote or counteract cell death, onset of tumors and neurodegenerative diseases have been constructed. The effects on yeast cells of some of these genes are also presented

Hypothesis: is yeast a clock model to study the onset of humans aging phenotypes?

In this paper we report the growth and aging of yeast colonies derived from single cells isolated by micromanipulation and seeded one by one on separated plates to avoid growth interference by surrounding colonies. We named this procedure clonal life span, and it could represent a third way of studying aging together with the replicative life span and chronological life span. In this study we observed over time the formation of cell mass similar to the human "senile warts" (seborrheic keratoses), the skin lesions that often appear after 30 years of life and increase in number and size over the years. We observed that similar signs of aging appear in yeast colonies after about 27 days of growth and increase during aging. In this respect we hypothesize to use yeast as a clock to study the onset of human aging phenotypes

Use of the KlADH4 promoter for ethanol-dependent production of recombinant human serum albumine in Kluyveromyces lactis

KlADH4 is a gene of Kluyveromyces lactis encoding a mitochondrial alcohol dehydrogenase activity which is specifically induced by ethanol. The promoter of this gene was used for the expression of heterologous proteins in K. lactis, a very promising organism which can be used as an alternative host to Saccharomyces cerevisiae due to its good secretory properties. In this paper we report the ethanol-driven expression in K. lactis of the bacterial beta-glucuronidase and of the human serum albumin (HSA) genes under the control of the KlADH4 promoter. In particular, we studied the extracellular production of recombinant HSA (rHSA) with integrative and replicative vectors and obtained a significant increase in the amount of the protein with multicopy vectors, showing that no limitation of KlADH4 trans-acting factors occurred in the cells. By deletion analysis of the promoter, we identified an element (UASE) which is sufficient for the induction of KlADH4 by ethanol and, when inserted in the respective promoters, allows ethanol-dependent activation of other yeast genes, such as PGK and LAC4. We also analyzed the effect of medium composition on cell growth and protein secretion. A clear improvement in the production of the recombinant protein was achieved by shifting from batch cultures (0.3 g/liter) to fed-batch cultures (1 g/liter) with ethanol as the preferred carbon source

The control of ovarian activity in gilt and the effect of progesteron and prostaglandin administration on oestrus synchronization

Lo scopo di questa tesi è stato quello di valutare l’effetto della somministrazione del progesterone e delle prostaglandine, sulla sincronizzazione degli estri nella scrofetta. Nel primo studio (Esperimento 1), è stato valutato se la doppia somministrazione a livello perivulvare nella scrofetta, di una dose elevata di cloprostenolo (analogo di sintesi delle PGF2alfa) fosse in grado di indurre una luteolisi al 7°, 9° o 10° gg del ciclo estrale. Per questo scopo 87 scrofette puberi sono state trattate con una doppia (ore 08:00 e 14:00) somministrazione di 75 µg D-cloprostenol al 7° (gruppo D7, n°=30), 9° (groppo D9; n°=29) e 10° (gruppo D10; n°=28) giorno del ciclo estrale. La durata del ciclo estrale durante il quale è stato effettuato il trattamento è stata confrontata con la durata del ciclo estrale precedente al trattamento. Il profilo ematico del progesterone è stato determinato in dieci scrofette del gruppo D9. In tutti e tre i gruppi la durata del ciclo estrale con il trattamento non è stata diversa dal ciclo estrale precedente al trattamento (tra i 18 ed i 19 gg). I livelli plasmatici di progesterone dopo il trattamento si sono ridotti in 7/10 strofette ma solo in 3 scrofette si sono mantenuti bassi 72 ore dopo il trattamento (intorno ai 1.3±0.9 ng/ml). I risultati di questo studio indicano che la doppia somministrazione di D-Cloprostenolo a livello perivulvare al 7°, 9° o 10° gg del ciclo estrale non induce una luteolisi completa ma solo parziale e la durata del ciclo estrale non viene modificata. Nel secondo esperimento è stata messa a punto una tecnica di cateterizzazione permanente per effettuare dei prelievi ematici frequenti nella specie suina per diversi giorni, senza richiedere un intervento chirurgico e anestetico, ma solo il contenimento dell’animale. Come sede sono state scelte le vene auricolari. I cateteri utilizzati sono stati il Dog Catheter (PVC IV 1.3x 300 mm-4FG) (n°=10 scrofe) ed il Polyetilene Tubing (I.D 0.76 mm (0.030” pollici ) O.D. 1.22 (0.048”) (n°=10 scrofe). Il catetere, è stato inserito nella vena auricolare mediale o laterale fino alla vena cava in prossimità del cuore mediante tecnica “through the needle”. I prelievi sono stati eseguiti ogni 15 min. dalle ore 9.00 alle ore 15.00 o fino a quando il catetere è rimasto funzionale o fino ad un massimo di 10 giorni consecutivi. Il tempo medio richiesto per la cateterizzazione con il Polyetilene Tubing è stato di 16-17 min. ed è stato possibile eseguire i prelievi per 8-9 giorni consecutivi, mentre la cateterizzazione con il Dog Catheter ha richiesto 21-23 minuti ed è rimasta funzionale per 4-5 giorni. I risultati indicano che il catetere inserito nella vena auricolare rimane in situ per diversi giorni (8-9) permettendo di eseguire dei prelievi frequenti con uno stress minimo per l’animale essenzialmente dovuto al contenimento dello stesso al momento dell’inserimento del catetere. Nel terzo esperimento è stato sincronizzato l'estro nelle scrofette, mediante la somministrazione di 20 mg/giorno di Alliltrembolone per via orale per 18, 14, 12 e 10 giorni dove, in questi ultimi due gruppi, il progestinico è stato seguito dalla somministrazione di una doppia dose di 75 µg D-cloprostenolo. Non sono stati riscontrati effetti della durata del trattamento sulla sincronizzazione dei parti, sul numero di scrofette in estro, sul numero dei parti o sulle dimensioni della nidiata. In fine l’obiettivo dell’ultimo esperimento, è stato quello di determinare se l’incidenza dei ritorni in estro regolari o irregolari mostrasse variazioni nel corso dei vari mesi dell’anno nel Nord Italia. In tale studio 5.103 scrofe, tornate in estro dopo l’inseminazione effettuata dopo lo svezzamento, sono state suddivise in differenti gruppi in base alla durata del loro intervallo inter-estrale: - Ritorno in estro regolare di tipo 1 (RR-1): scrofe che sono tornate in estro 18-23 giorni dopo l’inseminazione. - Ritorno in estro regolare di tipo 2 (RR-2): scrofe che sono tornate in estro 36-48 giorni dopo l’inseminazione. - Ritorno in calore irregolare (gruppo RI): scrofe che sono ternate in estro a 24-35 giorni dopo l’inseminazione. Sulla base delle temperature stagionali si è osservata una prevalenza (P<0.05) dei ritorni di tipo RR durante il periodo di Giugno-Agosto mentre durante i mesi di Settembre e Ottobre si è osservata una prevalenza dei ritorni di tipo RI. I risultati di questo studio indicano che, nel Nord Italia, durante i mesi più caldi dell’anno, ci sia un incremento di ritorni in estro regolari, indicando un problema nell’efficacia dell’inseminazione, viceversa, durante il periodo di Settembre e Ottobre, aumentano ritorni in estro irregolari, indicando una maggior prevalenza di perdite embrionali.The aim of this thesis has been to investigate the effect of progesterone and prostaglandin administration, on oestrus synchronization in gilt. In the experiment 1. Has been evaluated if a double, perivulval large dose of PGF2alfa can induce luteolysis before day 12 of oestrus cycle in gilts. Following a pre-treatment oestrous cycle, 87 gilts were assigned to receive vulva injections of 75 µg D-cloprostenol at 08:00 and 14:00 h on day 7 (group D7; n=30), day 9 (group D9; n=29) or day 10 (group D10; n=28) of their second observed oestrous cycle. Comparisons were made between the previous control and the following treatment oestrous cycles. Ten D9 gilts were blood sampled 6 h prior to first injection and at 24 and 72 h after the second injection and samples assayed for progesterone content. Compared to pre-treatment levels (15.8±0.7 ng/ml), plasma progesterone concentrations were reduced (p<0.05) at 24 h (6.4±0.4 ng/ml) in 7 of 10 gilts. However, at 72 h serum progesterone had returned to pre-treatment levels in 4 of the 7 gilts while in the remaining 3 gilts serum progesterone concentrations continued to fall to 1.3±0.9 ng/ml. Across all treatments, the duration of the treatment oestrous cycle was not affected by D-cloprostenol administration. These data indicate that swine corpora lutea are sensitive to prostaglandins from day 9 of the oestrous cycle but that a terminal luteolysis usually does not ensue. In the second experiment, has been utilized a new technique for chronic cannulation in gilt to allow frequent samplings. For this study 20 pluriparous sows have been used. The technique involved the implantation of a catheter, with a “through the needle” technique, via the lateral or medial ear vein. The adopted catheters were: Dog Catheter (PVC IV 1.3x 300 mm-4FG) (n°=10 sows) and Polyetilene Tubing I.D 0.76 mm (0.030” inch) O.D. 1.22 (0.048”) 90-120 cm, (n°=10 sows). Frequent blood samplings have been performed every 15 minutes for 6h consecutively from 9 a.m. to 3 p.m. for 10 days consecutively or until the catheter was functional. The ear vein catheterization with Polyetilene Tubing has been performed in 16-17 minutes and remained patent for 8-9 days, while the catheterization with the Dog Catheter required 21-23 minutes and remained patent for 4-5 days. During the trial the pig behaved normally, presented a good appetite and normal body temperature. The permanent ear venous cannulation enables to collect sequential blood samples for several days with minimum restraint of the animal, it does not require surgical procedures under general anaesthesia and can be applied routinely in the farm. In experiment three to synchronize oestrus in gilts, was administrated Allyltrenbolone at 20 mg per day for 10, 12, 14, or 18 days, with injection of 2×75 µg D-cloprostenol at last feeding at 10 or 12 days. There were no treatment effects on farrowing rate or subsequent litter sizes. Finally in the last experiment (experiment 4), was determined the incidence of regular or irregular inter-oestrus interval in sows during different months of the year in Northern Italy. In this study, 5.103 sows that returned to oestrus were inseminated and assigned to different groups based on the length of their inter-oestrus interval: - Regular inter-oestrus interval type 1 (RR-1): sows with inter-oestrus interval of 18-23 days - Regular inter-oestrus interval type 2 (RR-2): sows with inter-oestrus interval of 36-48 days - Irregular inter-oestrus interval (IR group): inter-oestrus interval of 24-35. Based on meteorological data of the area in which the farms were located, animals were considered to be under heat stress during the high temperature months of June to August while animals were not considered to be under heat stress during September to May. The proportion of sows with RR-1 was greater (P<0.05) during the high temperature month compared than during the rest of the year. There was no difference between months for the proportion of sows with RR-2. The proportion of sows with IR was greater (P<0.05) during the months of September and October than during the other months of the year. Results from this study indicate that, in Northern Italy, during the high temperature months of June-August, there is an increase in the proportion of sows with regular inter-oestrus interval, suggesting a failure of conception or an increase of early embryos losses, while there is an increase in the proportion of sows with irregular inter-oestrus interval during September-October, suggesting later embryo losses

Increased levels of RNA oxidation enhance the reversion frequency in aging pro-apoptotic yeast mutants

Despite recent advances in understanding the complexity of RNA processes, regulation of the metabolism of oxidized cellular RNAs and the mechanisms through which oxidized ribonucleotides affect mRNA translation, and consequently cell viability, are not well characterized. We show here that the level of oxidized RNAs is markedly increased in a yeast decapping Kllsm4Δ1 mutant, which accumulates mRNAs, ages much faster that the wild type strain and undergoes regulated-cell-death. We also found that in Kllsm4Δ1 cells the mutation rate increases during chronological life span indicating that the capacity to han- dle oxidized RNAs in yeast declines with aging. Lowering intracellular ROS levels by antioxidants recovers the wild- type phenotype of mutant cells, including reduced amount of oxidized RNAs and lower mutation rate. Since mRNA oxidation was reported to occur in different neurodegen- erative diseases, decapping-deficient cells may represent a useful tool for deciphering molecular mechanisms of cell response to such conditions, providing new insights into RNA modification-based pathogenesis

Benchmarking of pluck lesions at slaughter as a health monitoring tool for pigs slaughtered at 170 kg (heavy pigs)

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Superficial and deep changes of histology, texture and particle size distribution in broiler wooden breast muscle during refrigerated storage

Abstract Recently the poultry industry faced an emerging muscle abnormality termed wooden breast (WB), the prevalence of which has dramatically increased in the past few years. Considering the incomplete knowledge concerning this condition and the lack of information on possible variations due to the intra-fillet sampling locations (superficial vs. deep position) and aging of the samples, this study aimed at investigating the effect of 7-d storage of broiler breast muscles on histology, texture, and particle size distribution, evaluating whether the sampling position exerts a relevant role in determining the main features of WB. With regard to the histological observations, severe myodegeneration accompanied by accumulation of connective tissue was observed within the WB cases, irrespective of the intra-fillet sampling position. No changes in the histological traits took place during the aging in either the normal or the WB samples. As to textural traits, although a progressive tenderization process took place during storage (P ≤ 0.001), the differences among the groups were mainly detected when raw meat rather than cooked was analyzed, with the WB samples exhibiting the highest (P ≤ 0.001) 80% compression values. In spite of the increased amount of connective tissue components in the WB cases, their thermally labile cross-links will account for the similar compression and shear-force values as normal breast cases when measured on cooked samples. Similarly, the enlargement of extracellular matrix and fibrosis might contribute in explaining the different fragmentation patterns observed between the superficial and the deep layer in the WB samples, with the superficial part exhibiting a higher amount of larger particles and an increase in particles with larger size during storage, compared to normal breasts

Orientus ishidae (Hemiptera: Cicadellidae): biology, direct damage and preliminary studies on apple proliferation infection in apple orchard

The mosaic leafhopper, Orientus ishidae (Matsumura), is an Asian species widespread in Europe that can cause leaf damage in wild trees and transmit disease phytoplasmas to grapevines. Following an O. ishidae outbreak reported in 2019 in an apple orchard in northern Italy, the biology and damage caused by this species to apples were investigated during 2020 and 2021. Our studies included observations on the O. ishidae life cycle, leaf symptoms associated to its trophic activity, and its capability to acquire "Candidatus Phytoplasma mali," a causal agent of Apple Proliferation (AP). The results indicate that O. ishidae can complete the life cycle on apple trees. Nymphs emerged between May and June, and adults were present from early July to late October, with the peak of flight between July and early August. Semi-field observations allowed for an accurate description of leaf symptoms that appeared as a distinct yellowing after a one-day exposure. In field experiments, 23% of the leaves were found damaged. In addition, 16-18% of the collected leafhoppers were found carrying AP phytoplasma. We conclude that O. ishidae has the potential to be a new apple tree pest. However, further studies are required to better understand the economic impact of the infestations

Assisted code point positioning at sub-meter accuracy level with ionospheric corrections estimated in a local GNSS permanent network

It is well know that GNSS permanent networks for real-time positioning with stations spaced at few tens of kilometers in the average were mainly designed to generate and transmit products for RTK (or Network-RTK) positioning. In this context, RTK products are restricted to users equipped with geodetic-class receivers which are continuously linked to the network processing center through Internet plus mobile phone. This work is a first step toward using a local network of permanent GNSS stations to generate and make available products devoted to ionospheric delay correction that could remarkably improve positioning accuracy for C/A receiver users, without forcing them to keep a continuous link with the network. A simple experiment was carried out based on data from the RESNAP-GPS network (w3.uniroma1.it/resnap-gps), located in the Lazio Region (Central Italy) and managed by DITS-Area di Geodesia e Geomatica, University of Rome “Sapienza”. C/A raw observations were processed with Bernese 5.0 CODSPP module (single point positioning based on code measurements) using IGS precise ephemeris and clocks. Further, the RINEX files were corrected for the Differential Code Biases (DCBs) according to IGS recommendations. One position per epoch (every 30 s) was estimated from C/A code; the vertical coordinate errors showed a typical signature due to the ionospheric activity: higher errors for day-time (up to 5 m) and smaller ones for night-time (around 1.5 m). In order to improve the accuracy of the solution, ionospheric corrections were estimated using the La Plata Ionospheric Model, based on the dual-frequency observations from the RESNAP-GPS network. This procedure allowed to reduce horizontal and vertical errors within 0.5 m (CE95) and 1 m (LE95) respectively. Finally, the possibility to predict the ionospheric model for few hours was preliminary checked. Our approach shows the possibility of a novel use of the measurements collected by GNSS permanent networks designed for real-time positioning services, which can assist and remarkably improve the C/A code real-time positioning supplying off-line predicted ionospheric corrections, acting as a local Ground Based Augmentation System.Facultad de Ciencias Astronómicas y Geofísica

Double cloprostenol administration during mid luteal phase of oestrous cycle does not modify the interoestrous interval in gilts

The present study was undertaken to test the effect of two vulva injections of D-cloprostenol on day 7, 9 and 10 of oestrous cycle on the duration of the interestrous interval in gilts. Following a pre-treatment oestrous cycle, 87 gilts were assigned to receive vulva injections of 75 μg D-cloprostenol at 08:00 and 14:00 h on day 7 (D7; n=30), day 9 (D9; n=29) or day 10 (D10; n=28) of their second observed oestrous cycle. Across the treatments, the duration of the oestrous cycle with D-cloprostenol treatment (19.1±0.1 d) was not different from that of the previous oestrous cycle (20.1±0.4 days). Plasma progesterone concentrations were evaluated 6 h before and 24 and 72 h after D-cloprostenol treatment in the D9 group. Compared to pretreatment levels (9.6±0.4 ng/mL), plasma progesterone concentrations were reduced (P<0.05) at 24 h (6.3±1.0 ng/mL) and 72 h after treatment but complete luteolysis did not occur. These data indicate that in gilts double vulva administration of D-cloprostenol is not able to induce a complete luteolisys and hence the duration of the oestrous cycle is not modified