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Recent experimental studies have suggested that an increase in the synthesis of nitric oxide, a powerful vasodilator secreted by endothelial cells, plays a role in the hemodynamic disturbances associated to portal hypertension. The present study was addressed to investigate the effects of L-NNA (a specific inhibitor of nitric oxide) on systemic and splanchnic hemodynamics in portal hypertensive rats, induced by partial portal vein ligation. Intravenous infusion of L-NNA (50 ug/kg/min) significantly increased systemic blood pressure and decreased cardiac output as measured by radiolabeled microspheres. A significant increase in systemic and splanchnic vascular resistance was also observed in L-NNA-treated rats; whereas portal blood flow decreased significantly, L-NNA did not modify portal pressure. Pretreatment with L-arginine (300 mg/Kg, i.v.) prevented the hemodynamic changes induced by L-NNA. Similar values of endotoxin levels were detected in both groups of animals. In the control group, L-NNA caused a mild but significant increase of mean arterial pressure; no significant changes on the other hemodynamic parameters were observed. These results suggest that an increase in endogenous synthesis of nitric oxide may play an important role in hemodynamic disturbances associated with chronic portal hypertension.

In this work we have demonstrated the influence of a short chain fatty acid (acetate) on the number of enterochromaffin (EC) cells containing serotonin (5HT), at two different pH (pH 6.9 absorptive stimuli, and pH 2.9 secretory stimuli), infused into the colon during one hour. The number of EC cells decrease significatively, specially in the cecum with a solution of low pH (2.9). The action of piprenzepine in preventing this reduction demonstrated that was partly mediated by a cholinergic receptor mechanism. On the other hand, a decrease on the release of 5HT to the lumen was a observed under the influence of pirenzepine. We conclude that the short chain fatty acid acetate, at a low pH induces the release of serotonin through a cholinergic mechanisms indicated by the inhibition observed with antimuscarinic drug.

Recently, 5 sibling species and 3 other phenotypes were identified in the genus Trichinella. Single primers of arbitrary nucleotide sequence were used to produce random amplified polymorphic DNA starting from decreasing amounts of Trichinella spiralis and Trichinella britovi DNA. Reproducible amplification products from 30 pg of DNA were obtained using 1 of 6 examined primers. These fragments distinguish between 2 European Trichinella species, T. spiralis, showing a 1,350-bp band, and T. britovi, showing 400- and 1,100-bp bands. The developed procedure allows the characterization of crude DNA preparations of single muscle-stage larvae, avoiding time-consuming passages of parasites in laboratory animals.

In vitro amplification of genomic DNA fragments with single primers of arbitrary sequence was used as a rapid and sensitive method to obtain fingerprints of ten strains belonging to three of the Azospirillum species: brasilense, lipoferum and amazonense. Each strain showed a distinctive pattern of bands that permitted its unequivocal identification. Closely related strains produced almost identical fingerprints. Pairwise comparison and cluster analysis of the amplification patterns allowed grouping of the strains. The resulting dendrograms are similar to previous dendrograms based on the restriction endonuclease analysis (REA) of total DNA and on the restriction fragment length polymorphism (RFLP). Our results indicate that the random amplified polymorphic DNA (RAPD) technique is a simple, fast and useful tool for the determination of genetic relationships among Azospirillum isolates and to evaluate the genomic stability of the Azospirillum strains released in the environment.

The possibility that VIP (Vasoactive intestinal peptide) could influence the enterochromaffin (EC) cell secretion of serotonin (5HT) and the action of VIP on the mast cell population of lamina propria were investigated in Wistar rat colon infused with a short chain fatty acid solution (sodium acetate), during a 1 h period. Under the action of an intravenous injection of synthetic porcine VIP, 14 micrograms/kg/h), the number of EC cells diminished significantly in the cecum and left colon, when compared to non-injected animals, both infused with a sodium acetate solution. At the same time, the number of mucosal mast cells in the crypts and lamina propria decreased significantly in the cecum. The postulate we put forward is that these VIP-induced changes are exerted through the stimulation of 5HT released from EC cells not only under normal physiological conditions but probably also under pathological conditions.

We analyzed the effect of drugs that modify the colonic motility on rat intestinal transit time (ITT) (measured with radiopaque markers), fecal weight (FW) and fecal concentration of short chain fatty acids (FSCFA) (assayed by gas liquid chromatography), over a four-week period. Bisacodyl was used to accelerate and Loperamide to retard the intestinal transit in rats maintained on a conventional diet. The first and 3rd week were drug-free control periods. The mean values of these periods were: ITT = 28.9h +/- 1.9 FW: 9.2 +/- 1.2 g/24 h and FSCFA = 60.6 +/- 17.9 mmol/g. After loperamide administration, we observed an increase in the mean ITT as compared to the control period (40.4 +/- 8.0h) and decrease in FW (4.8 +/- 3.6 g/24h) and in FSCFA = 32.2 +/- 5.6 mmol/g). After bisacodyl administration, we found a shorter ITT in relation to controls (24.8 +/- 2.5h), and increases in FW (27.5 +/- 3.6g/24h) and in FSCFA (108.2 +/- 39.9 mmol/g). There was a negative correlation between ITT and FW (R = 0.67 p less than 0.01) and a positive correlation between total SCFA concentration and FW (R = 0.71 p less than 0.01). The concentration of acetic, propionic and butyric acids increased with progressive increments in fecal weight, whereas concentrations of isovaleric and caproic acids decreased. The results of this study show that the FW and the FSCFA may be influenced by modifications in the intestinal transit time.

Chromatographic methods, especially thin-layer chromatography (TLC) and gas-liquid chromatography (GLC) are widely used in investigations of the occurrence, molecular structure and metabolism of ether lipids. The application of such techniques to structural analysis and quantification, in combination with methods for the degradation and derivatization of ether lipids, is discussed.

1-O-[1'-14C]Hexadecyl ethanediol was administered intracerebrally to myelinating rat brain, and incorporation of radioactivity into brain lipids was followed over a 48-h period: 1. (1) O-Hexadecyl ethanediol was metabolized primarily through oxidative ether bond cleavage, and much of the label was recovered in phospholipid acyl groups. 2. (2) Substantial amounts of radioactivity were also found in choline and ethanolamine phospholipids having an O-hexadecyloxyethyl glycerol backbone. This means that alkyl ethanediol was used in glycerol ether biosynthesis as are long-chain primary alcohols. 3. (3) Acidic hydrolysis of the ethanolamine glycerophosphatide fraction yielded also labeled hexadecanol which may indicate desaturation of 1-O-hexadecyloxyethyl 2-acyl glycerophosphoryl ethanolamine to the plasm alogen analogue. 4. (4) Small amounts of the substrate were oxidized to O-hexadecyl glycolic acid and incorporated into the phospholipids. The substrate did not serve as precursor of O-hexadecyl ethanediol phosphorylcholine or phosphorylethanolamine in the brain.

Long-chain 1,2-alkanediols can be separated into their enantiomers by thin-layer chromatography of the bis-L-acetylmandelates. The method is also applicable to the separation of enantiomeric 1,3-alkanediols. Both 2-hydroxy fatty acids and 1,2-alkanediols derived from diester waxes of rat skin have the D-configuration. The D-enantiomer of 2DL-1,2-octadecanediol is preferentially utilized by myelinating rat brain in the formation of choline phospholipids having a 1,2-octadecanediol backbone. The D-configuration of the chiral center at carbon-2 of 2-acyl-1,2-alkanediol phosphocholine corresponds to the configuration at carbon-2 of 1,2-diacyl-sn-glycero-3-phosphocholine.

1- O -[1'- 14 C ]Hexadecyloxyethyl rac -glycerol was administered to 18-day-old rats by intracerebral injection, and incorporation of radioactivity into the brain lipids was determined after 6, 24 and 48 h. Some of the substrate was catabolized by oxidative cleavage of either of the two ether bonds. Cleavage in the hexadecyloxyethyl moiety yielded labeled palmitic acid, whereas oxidative cleavage of the glycol glycerol ether bond produced O -hexadecyl glycolic acid. The substrate was also incorporated as such into both ethanolamine and choline phospholipids. Evidence is presented for the desaturation by rat brain of 1- O -hexadecyloxyethyl-2-acyl- sn -glycero-3-phosphoethanolamine to the plasmalogen analogue, while the corresponding choline phospholipid was not desaturated.

Summary Microsomal preparations from rat liver catalyze the formation of 2-acyl-1,2-alkanediolphosphorylcholine from a long-chain 2-acyl-1,2-alkanediol and CDP-choline in the presence of Mg ++ or Mn ++ . The reaction is inhibited by Ca ++ . The results indicate that 2-acyl-1,2-alkanediol can substitute for 1,2-diglyceride in phospholipid biosynthesis.

1- O -[1′-14C]Hexadecyl ethanediol 2-phosphorylethanolamine was administered to myelinating rat brain in order to establish general patterns of diol phospholipid metabolism and the specific substrate requirements of 1- O -alkyl 2-acyl sn -glycero-3-phosphorylethanolamine desaturase. It was shown that alkyl ethanediol PE was slowly metabolized by oxidative ether bond cleavage. Dehydrogenation to diol plasmalogen did not occur, thus providing further evidence for the high degree of substrate specificity of the desaturase present in brain.

1-O-[1'-14C]Hexadecyl ethanediol was administered intracerebrally to myelinating rat brain, and incorporation of radioactivity into brain lipids was followed over a 48-h period: (1) O-Hexadecyl ethanediol was metabolized primarily through oxidative ether bond cleavage, and much of the label was recovered in phospholipid acyl groups. (2) Substantial amounts of radioactivity were also found in choline and ethanolamine phospholipids having an O-hexadecyloxyethyl glycerol backbone. This means that alkyl ethanediol was used in glycerol ether biosynthesis as are long-chain primary alcohols. (3) Acidic hydrolysis of the ethanolamine glycerophosphatide fraction yielded also labeled hexadecanol which may indicate desaturation of 1-O-hexadecyloxyethyl 2-acyl glycerophosphoryl ethanolamine to the plasmalogen analogue. (4) Small amounts of the substrate were oxidized to O-hexadecyl glycolic acid and incorporated into the phospholipids. The substrate did not serve as precursor of O-hexadecyl ethanediol phosphorylcholine or phosphorylethanolamine in the brain.

A fraction comprising about 0.7% of the extractable surface lipids of cabbage (Brassica oleracea) leaves was isolated and identified as a mixture of isomeric beta-hydroxyketones consisting mainly of 14-keto-16-hydroxynonacosane and 15-keto-13-hydroxynonacosane.

The monounsaturated alk-I-enyl, alkyl and acyl moieties in the lipids of ratfish ( Hydrolagus colliei ) liver comprise mixtures of positional isomers. The major alk-I-enyl chain of the O -alk-I-enyl diglycerides has the structure Δ 13–20:1; the predominant alkyl chain of the O -alkyl diglycerides has the structure Δ9–18 : I In all three lipid classes, the structure of the major acyl chain is Δ9–18:1. Similarities between the positional isomers of the alkyl and acyi moieties strongly suggest a close metabolic relationship between alkyl ethers and esters in ratfish liver. However, the alk-I-enyl chains differ markedly from both the alkyl and acyl moieties in chain lengths and in position of double bonds.

Background & Aims Information about the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in patients with liver cancer is lacking. This study characterizes the outcomes and mortality risk in this population. Methods Multicentre retrospective, cross-sectional, international study of liver cancer patients with SARS-CoV-2 infection registered between February and December 2020. Clinical data at SARS-CoV-2 diagnosis and outcomes were registered. Results Two hundred fifty patients from 38 centres were included, 218 with hepatocellular carcinoma (HCC) and 32 with intrahepatic cholangiocarcinoma (iCCA). The median age was 66.5 and 64.5 years, and 84.9% and 21.9% had cirrhosis in the HCC and iCCA cohorts respectively. Patients had advanced cancer stage at SARS-CoV-2 diagnosis in 39.0% of the HCC and 71.9% of the iCCA patients. After a median follow-up of 7.20 (IQR: 1.84–11.24) months, 100 (40%) patients have died, 48% of the deaths were SARS-CoV-2-related. Forty (18.4%) HCC patients died within 30-days. The death rate increase was significantly different according to the BCLC stage (6.10% [95% CI 2.24–12.74], 11.76% [95% CI 4.73–22.30], 20.69% [95% CI 11.35–31.96] and 34.52% [95% CI 17.03–52.78] for BCLC 0/A, B, C and D, respectively; p = .0017). The hazard ratio was 1.45 (95% CI 0.49–4.31; p = .5032) in BCLC-B versus 0/A, and 3.13 (95% CI 1.29–7.62; p = .0118) in BCLC-C versus 0/A in the competing risk Cox regression model. Nineteen out of 32 iCCA (59.4%) died, and 12 deaths were related to SARS-CoV-2 infection. Conclusions This is the largest cohort of liver cancer patients infected with SARS-CoV-2. It characterizes the 30-day mortality risk of SARS-CoV-2 infected patients with HCC during this period., Spanish Health Ministry; Ricerca Corrente, Grant/Award Number: RC2019/105-01, RC2018/105-01 and RC2017/105-01; European Association for the Study of the Liver (Andrew Burroughs Fellowship); Cancer Treatment and Research Trust (CTRT); Wellcome Trust Strategic Fund, Grant/Award Number: PS3416; Cancer Research UK (CR UK) centre, Grant/Award Number: C9380/A26813, C18342/A23390 and C9380/A18084; Instituto de Salud Carlos III, Grant/Award Number: PI18/0358, PI15/00145, CD18/00126, PI19/00819, FI19/00222, 16-0026, PI044031, PI18/00768, PI18/00542 and PI13/01229; Bristol Myers Squibb and Celgene; Cancer Research UK, Grant/Award Number: RCCPDB-Nov21/100008

Since a few decades, apiculture is facing important economic losses worldwide with general major consequences in many areas of agriculture. A strong attention has been paid towards the phenomenon named Colony Collapse Disorder in which colonies suddenly disappear with no clear explanations. Honeybee colonies can be affected by abiotic factors, such as environmental pollution or insecticide applications for agricultural purposes. Also biotic stresses cause colony losses, including bacterial (e. g. Paenibacillus larvae) and fungal (e. g. Ascosphaera apis) pathogens, microsporidia (e. g. Nosema apis), parasites (i.e. Varroa destructor) and several viruses. In the light of recent research, intestinal dysbiosis, considered as the relative disproportion of the species within the native microbiota, has shown to affect human and animal health. In arthropods, alteration of the gut microbial climax community has been shown to be linked to health and fitness disequilibrium, like in the medfly Ceratitis capitata for which low mate competitiveness is determined by a gut microbial community imbalance. According to these observations, it is possible to hypothesize that dysbiosis may have a role in disease occurrence also in honeybees. Here we aim to discuss the current knowledge on dysbiosis in the honeybee and its relation with honeybee health by reviewing the investigations of the microbial diversity associated to honeybees and the recent experiments performed to control bee diseases by microbial symbionts. We conclude that, despite the importance of a good functionality of the associated microbiota in preserving insect health has been proved, the mechanisms involved in honeybee gut dysbiosis are still unknown. Accurate in vitro, in vivo and in field investigations are required under healthy, diseased and stressed conditions for the host

Summary Bacterial symbionts of insects have been proposed for blocking transmission of vector-borne pathogens. However, in many vector models the ecology of sym- bionts and their capability of cross-colonizing differ- ent hosts, an important feature in the symbiotic control approach, is poorly known. Here we show that the acetic acid bacterium Asaia, previously found in the malaria mosquito vector Anopheles stephensi, is also present in, and capable of cross-colonizing other sugar-feeding insects of phylogenetically distant genera and orders. PCR, real-time PCR and in situ hybridization experiments showed Asaia in the body of the mosquito Aedes aegypti and the leafhopper Scaphoideus titanus, vectors of human viruses and a grapevine phytoplasma respectively. Cross- colonization patterns of the body of Ae. aegypti, An. stephensi and S. titanus have been documented with Asaia strains isolated from An. stephensi or Ae. aegypti, and labelled with plasmid- or chromosome-encoded fluorescent proteins (Gfp and DsRed respectively). Fluorescence and confocal microscopy showed that Asaia, administered with the sugar meal, efficiently colonized guts, male and female reproductive systems and the salivary glands. The ability in cross-colonizing insects of phylogenetically distant orders indicated that Asaia adopts body inva- sion mechanisms independent from host-specific bio- logical characteristics. This versatility is an important property for the development of symbiont-based control of different vector-borne diseases.

SUMMARYThe hard tickIxodes ricinus(Ixodidae) is the sole animal thus far shown to harbour an intra-mitochondrial bacterium, which has recently been namedMidichloria mitochondrii. The objectives of this work were (i) to screen ixodid ticks forMidichloria-related bacteria and (ii) to determine whether these bacteria exploit the intra-mitochondrial niche in other tick species. Our main goal was to discover further models of this peculiar form of symbiosis. We have thus performed a PCR screening forMidichloria-related bacteria in samples of ixodid ticks collected in Italy, North America and Iceland. A total of 7 newly examined species from 5 genera were found positive for bacteria closely related toM. mitochondrii. Samples of the tick speciesRhipicephalus bursa, found positive in the PCR screening, were analysed with transmission electron microscopy, which revealed the presence of bacteria both in the cytoplasm and in the mitochondria of the oocytes. There is thus evidence that bacteria invade mitochondria in at least 2 tick species. Phylogenetic analysis on the bacterial 16S rRNA gene sequences generated from positive specimens revealed that the bacteria form a monophyletic group within the order Rickettsiales. The phylogeny ofMidichloriasymbionts and related bacteria does not appear completely congruent with the phylogeny of the hosts.

Here, we show that an α-proteobacterium of the genus Asaia is stably associated with larvae and adults of Anopheles stephensi , an important mosquito vector of Plasmodium vivax , a main malaria agent in Asia. Asaia bacteria dominate mosquito-associated microbiota, as shown by 16S rRNA gene abundance, quantitative PCR, transmission electron microscopy and in situ -hybridization of 16S rRNA genes. In adult mosquitoes, Asaia sp. is present in high population density in the female gut and in the male reproductive tract. Asaia sp. from An. stephensi has been cultured in cell-free media and then transformed with foreign DNA. A green fluorescent protein-tagged Asaia sp. strain effectively lodged in the female gut and salivary glands, sites that are crucial for Plasmodium sp. development and transmission. The larval gut and the male reproductive system were also colonized by the transformed Asaia sp. strain. As an efficient inducible colonizer of mosquitoes that transmit Plasmodium sp., Asaia sp. may be a candidate for malaria control.

Flavescence dorée (FD) is a grapevine disease that afflicts several wine production areas in Europe, from Portugal to Serbia. FD is caused by a bacterium, “ Candidatus Phytoplasma vitis,” which is spread throughout the vineyards by a leafhopper, Scaphoideus titanus (Cicadellidae). After collection of S. titanus specimens from FD-contaminated vineyards in three different areas in the Piedmont region of Italy, we performed a survey to characterize the bacterial microflora associated with this insect. Using length heterogeneity PCR with universal primers for bacteria we identified a major peak associated with almost all of the individuals examined (both males and females). Characterization by denaturing gradient gel electrophoresis confirmed the presence of a major band that, after sequencing, showed a 97 to 99% identity with Bacteroidetes symbionts of the “ Candidatus Cardinium hertigii” group. In addition, electron microscopy of tissues of S. titanus fed for 3 months on phytoplasma-infected grapevine plants showed bacterial cells with the typical morphology of “ Ca. Cardinium hertigii.” This endosymbiont, tentatively designated ST1-C, was found in the cytoplasm of previtellogenic and vitellogenic ovarian cells, in the follicle cells, and in the fat body and salivary glands. In addition, cell morphologies resembling those of “ Ca. Phytoplasma vitis” were detected in the midgut, and specific PCR assays indicated the presence of the phytoplasma in the gut, fat body and salivary glands. These results indicate that ST1-C and “ Ca. Phytoplasma vitis” have a complex life cycle in the body of S. titanus and are colocalized in different organs and tissues.