Neoechinulin A as a Promising SARS-CoV-2 Mpro Inhibitor: In Vitro and In Silico Study Showing the Ability of Simulations in Discerning Active from Inactive Enzyme Inhibitors

The COVID-19 pandemic and its continuing emerging variants emphasize the need to discover appropriate treatment, where vaccines alone have failed to show complete protection against the new variants of the virus. Therefore, treatment of the infected cases is critical. This paper discusses the bio-guided isolation of three indole diketopiperazine alkaloids, neoechinulin A (1), echinulin (2), and eurocristatine (3), from the Red Sea-derived Aspergillus fumigatus MR2012. Neoechinulin A (1) exhibited a potent inhibitory effect against SARS-CoV-2 M^pro with IC₅₀ value of 0.47 μM, which is comparable to the reference standard GC376. Despite the structural similarity between the three compounds, only 1 showed a promising effect. The mechanism of inhibition is discussed in light of a series of extensive molecular docking, classical and steered molecular dynamics simulation experiments. This paper sheds light on indole diketopiperazine alkaloids as a potential structural motif against SARS-CoV-2 M^pro. Additionally, it highlights the potential of different molecular docking and molecular dynamics simulation approaches in the discrimination between active and inactive structurally related M^pro inhibitors.     

Active circulation of bluetongue vaccine virus serotype-2 among unvaccinated cattle in central Italy

Several seroconversions occurring in 2002 among sentinel cattle during the bluetongue-vaccination campaign in Lazio and Tuscany (central Italy) led to the suspicion of vaccine-virus circulation. Therefore in 2003, 17 seroconverting sentinel herds were investigated for the characteristics of the virus involved. From these farms, 91 unvaccinated animals and 57 Culicoides pools were tested for the presence of the bluetongue vaccine virus (serotype-2) or other strains. The presence of vaccine virus serotype-2 was confirmed by PCR followed by restriction analysis in the whole blood of 17 unvaccinated sentinel cattle and 12 pools of Culicoides imicola or C. obsoletus. Of the 17 herds, five were positive only for vaccine virus serotype-2, four were positive for other strains and two for both the vaccine and other strains; the remaining premises were virologicaly negative. The vaccine virus serotype-2 also was detected in areas not included in the vaccination campaign.     

Protective effect of the AT137RQ and ARQK176 PrP alleles against classical scrapie in Sarda breed sheep

The susceptibility of sheep to scrapie is under the control of the host’s prion protein (PrP) gene and is also influenced by the strain of the agent. PrP polymorphisms at codons 136 (A/V), 154 (R/H) and 171 (Q/R/H) are the main determinants of susceptibility/resistance of sheep to classical scrapie. They are combined in four main variants of the wild-type ARQ allele: VRQ, AHQ, ARH and ARR. Breeding programmes have been undertaken on this basis in the European Union and the USA to increase the frequency of the resistant ARR allele in sheep populations. Herein, we report the results of a multi-flock study showing the protective effect of polymorphisms other than those at codons 136, 154 and 171 in Sarda breed sheep. All ARQ/ARQ affected sheep (n = 154) and 378 negative ARQ/ARQ controls from four scrapie outbreaks were submitted to sequencing of the PrP gene. The distribution of variations other than those at the standard three codons, between scrapie cases and negative controls, was statistically different in all flocks. In particular, the AT₁₃₇RQ and ARQK₁₇₆ alleles showed a clear protective effect. This is the first study demonstrating a protective influence of alleles other than ARR under field conditions. If further investigations in other sheep breeds and with other scrapie sources confirm these findings, the availability of various protective alleles in breeding programmes of sheep for scrapie resistance could be useful in breeds with a low frequency of the ARR allele and would allow maintaining a wider variability of the PrP gene.     

Paediatric HUS Cases Related to the Consumption of Raw Milk Sold by Vending Machines in Italy: Quantitative Risk Assessment Based on Escherichia coli O157 Official Controls over 7 years

A quantitative risk assessment (RA) was developed to estimate haemolytic‐uremic syndrome (HUS) cases in paediatric population associated with the consumption of raw milk sold in vending machines in Italy. The historical national evolution of raw milk consumption phenomenon since 2008, when consumer interest started to grow, and after 7 years of marketing adjustment, is outlined. Exposure assessment was based on the official Shiga toxin‐producing Escherichia coli O157:H7 (STEC) microbiological records of raw milk samples from vending machines monitored by the regional Veterinary Authorities from 2008 to 2014, microbial growth during storage, consumption frequency of raw milk, serving size, consumption preference and age of consumers. The differential risk considered milk handled under regulation conditions (4°C throughout all phases) and the worst time–temperature field handling conditions detected. In case of boiling milk before consumption, we assumed that the risk of HUS is fixed at zero. The model estimates clearly show that the public health significance of HUS cases due to raw milk STEC contamination depends on the current variability surrounding the risk profile of the food and the consumer behaviour has more impact than milk storage scenario. The estimated HUS cases predicted by our model are roughly in line with the effective STEC O157‐associated HUS cases notified in Italy only when the proportion of consumers not boiling milk before consumption is assumed to be 1%. Raw milk consumption remains a source of E. coli O157:H7 for humans, but its overall relevance is likely to have subsided and significant caution should be exerted for temporal, geographical and consumers behaviour analysis. Health education programmes and regulatory actions are required to educate people, primarily children, on other STEC sources.     

Microscale patchiness of nutrients in plankton communities

Autoradiography was used to identify the presence of nutrient patches produced by zooplankton. Algal cells which encounter patches of phosphorus-33 released by swimming animals accumulate more label than cells that do not enter the patches. Differential labeling of algae does not occur when turbulence in the fluid is increased by stirring. Nutrient patchiness at the scale of millimeters or less in nature probably influences the course of competition and coexistence among the phytoplankton.     

Morphological indices for canine spermatozoa based on the World Health Organization laboratory manual for human semen

Attempting to contribute to the development of a more objective morphological evaluation of dog spermatozoa, in this study the indices of multiple sperm defects (multiple abnormalities index [MAI]; teratozoospermic index [TZI]; sperm deformity index [SDI]) were calculated following the World Health Organization (WHO) guidelines. In Experiment I, the concordance of MAI, TZI and SDI with the proportions of morphologically normal spermatozoa (MNS) was evaluated in fresh ejaculated spermatozoa (dogs = 47). In Experiment II, the potential role of indices as prognostic values was assessed in spermatozoa of different origin and treatment (fresh ejaculated: n = 6; fresh epididymal: n = 6; frozen‐thawed ejaculated: n = 6) by their correlation with different semen parameters (motility, membrane integrity and acrosome status) and with an in vitro sperm function test. Samples with different proportions of MNS showed different values of SDI, the index that better represented the decline of sperm morphology in both fresh and frozen‐thawed samples (Exp. I and II; p < 0.05). No correlations between indices and semen parameters were observed (Exp. II), but when samples were evaluated collectively, negative correlations (SDI‐motility, p = 0.01; SDI‐acrosome integrity, p = 0.002) were found. Including all the defects of each spermatozoon, SDI might be a useful index during morphological analysis and better discriminates the increase in multiple defects. A more objective morphological evaluation for dog spermatozoa was achieved by the WHO method, and in vitro tests allowed to elucidate the validity of SDI as prognostic indicator of in vitro fertilizing potential.     

Definitive evidence for the actual contribution of yeast in the transformation of neutral precursors of grape aromas.

Experiments were designed to demonstrate the actual contribution of yeast in the formation of the primary aroma during the vinification of neutral grapes. Ruché was chosen as the model wine to study because of its unique fragrance. A yeast strain specific for Ruché was selected using a new and rapid isolation method for red wines. The results of this study can be summarized as follows: Skins from nonaromatic white or red grapes apparently contain most of the primary aroma compounds that are revealed in the must only after contact with yeast cells under defined conditions. Similar results were obtained with the pulp and seeds fractions; however, the olfactory notes, although well characterized, differed from those obtained with skins alone. Clarification, filtration, and centrifugation of the pulp and seed fractions or sonification of the skins produce different and well-characterized olfaction notes during the contact with yeast. The primary aroma of nonaromatic white and red grapes contained in the skins can be revealed within 24-48 h of yeast contact in a synthetic nutrient medium (SNM). The primary aroma precursors extracted from the skins with methanol, water-saturated butanol, or aqueous buffer at pH 3.2, concentrated and eluted from a C18 resin column, can be transformed to the free form wine aroma markers within 6 h of contact with yeast cells in SNM. By contrast, prolonged maceration of the skins in aqueous alcoholic buffer at pH 3.2 or 1.1, at 50 or 70 degrees C did not release primary odors typical of wine. The individual primary aroma compounds, identified by GC-MS analysis in Ruché wine samples or in Ruché skin-yeast-SNM samples, could not explain the complexity of the typical Ruché wine odor. Only odors common to many wine varieties were identified by GC-olfactometry analysis.