Griffithsin and Carrageenan Combination Results in Antiviral Synergy against SARS-CoV-1 and 2 in a Pseudoviral Model

Over 182 million confirmed cases of COVID-19 and more than 4 million deaths have been reported to date around the world. It is essential to identify broad-spectrum antiviral agents that may prevent or treat infections by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) but also by other coronaviruses that may jump the species barrier in the future. We evaluated the antiviral selectivity of griffithsin and sulfated and non-sulfated polysaccharides against SARS-CoV-1 and SARS-CoV-2 using a cytotoxicity assay and a cell-based pseudoviral model. The half-maximal cytotoxic concentration (CC₅₀) and half-maximal effective concentration (EC₅₀) were determined for each compound, using a dose-response-inhibition analysis on GraphPad Prism v9.0.2 software (San Diego, CA, USA). The therapeutic index (TI = CC₅₀/EC₅₀) was calculated for each compound. The potential synergistic, additive, or antagonistic effect of different compound combinations was determined by CalcuSyn v1 software (Biosoft, Cambridge, UK), which estimated the combination index (CI) values. Iota and lambda carrageenan showed the most potent antiviral activity (EC₅₀ between 3.2 and 7.5 µg/mL). Carrageenan and griffithsin combinations exhibited synergistic activity (EC₅₀ between 0.2 and 3.8 µg/mL; combination index <1), including against recent SARS-CoV-2 mutations. The griffithsin and carrageenan combination is a promising candidate to prevent or treat infections by SARS-CoV-1 and SARS-CoV-2.     

Molecular responses differ between sensitive silver carp and tolerant bighead carp and bigmouth buffalo exposed to rotenone

Some species of fish are more tolerant of rotenone, a commonly used non-specific piscicide, than others. This species-specific tolerance to rotenone has been thought to be associated with the uptake and the efficiency at which the chemical is detoxified. However, rotenone stimulates oxidative stress and superoxides, which are also toxic. Understanding the modes in which fish physiologically respond to rotenone is important in developing improved protocols for its application in controlling aquatic nuisance species. Using a molecular approach, we investigated the physiological and molecular mechanisms of rotenone resistance. Species-specific responses were observed when rotenone-sensitive silver, Hypophthalmichthys molitrix, and both rotenone-resistant bighead carp, Hypophthalmichthys nobilis, and bigmouth buffalo, Ictiobus cyprinellus, were exposed to rotenone. Rotenone levels in plasma were highest 90?min after exposure in both silver carp and bigmouth buffalo, but bigmouth buffalo tolerated over twice the burden (ng?mL(-1)?g(-1)) than silver carp. Expression of genes related with detoxification (cyp1a and gst) increased in silver carp, but either decreased or remained the same in bighead carp. Genes linked with oxidative stress in the cytosol (gpx, cat and sod1) and hsp70 increased only in silver carp after a 6-h exposure. Expression of genes associated with oxidative stress in the mitochondria (sod2 and ucp2) differed between silver carp and bighead carp. Expression of sod2 changed minimally in bighead carp, but expression of ucp2 linearly increased to nearly 85-fold of the level prior to exposure. Expression of sod2 and ucp2 did not change until 6?h in silver carp. Use of sod1 and sod2 to combat oxidative stress results in hydrogen peroxide production, while use of ucp2 produces nitric oxide, a chemical known to inhibit apoptosis. We conclude that the mechanism at which a fish handles oxidative stress plays an important role in the tolerance to rotenone.     

Shotgun proteomics of homogenate milk reveals dynamic changes in protein abundances between colostrum,transitional, and mature milk of swine

Milk is an easily digestible source of nutrients and bioactive factors, its composition reflects the neonate’s needs, and changes from colostrum to transitional and mature milk. Our objective was to measure milk fat, lactose, total carbohydrate, and protein content in parallel with global proteome of homogenate milk samples to characterize changes across the three phases of swine lactation. Milk samples were collected from multiparous sows (n = 9) on postnatal day 0 (D0; colostrum), 3 (D3; early transitional), 7 (D7; late transitional), and 14 (D14; mature). On D3, percent fat (16 ± 2.1) and lactose (3.8 ± 0.3) were higher (P < 0.05) than on D0 (10 ± 3.9 and 1.5 ± 0.3, respectively). Levels of fat and lactose were not different between D3 and D14. Percent total protein decreased (P < 0.05) between D0 (11 ± 2.1) and D3 (5 ± 0.7), but there was no significant change in percent protein between D3 and D14. Total carbohydrates increased (P < 0.05) between D3 (944 ± 353 µg/mL) and D14 (1,150 ± 462 µg/mL). Quantitative proteomic analysis using liquid chromatography tandem mass spectrometry (LC-MS/MS) of homogenate D0, D3, and D14 milk samples (n = 6) identified 772 protein groups which corresponded to 501 individual protein-coding genes. A total of 207 high confidence proteins were detected in n = 3 sows/day. Of the high confidence proteins, 81 proteins were common among all 3 days of lactation. Among the proteins that decreased between the days (false discovery rate; FDR < 0.05) were multiple apolipoproteins and XDH which decreased between D0 to D3. Proteins that increased across the days (FDR < 0.05) were complement factors and 14-3-3 proteins (YWHAQ, YWHAE). Our data provide a good characterization of milk proteome changes that likely reflect mammary function as well as the neonate’s phase-specific developmental needs. This data may be useful in developing approaches to enhance the health and welfare of swine.     

Improving the collection and germination of West African <Emphasis Type="Italic">Garcinia kola</Emphasis> Heckel seeds

Garcinia kola is highly used as chew-stick for dental care in West Africa, but it is nearly commercially extinct in nature. To help find methods to increase rate and percentage of germination, we investigated its seed germination responses by using several collections and pre-treatments. Seeds processed from 6-week old green fruits did not germinate, whilst those of 10-week old partly-mature, at the intermediate stage through full development, germinated 46%. Seeds from orange mature fruits reached maximum 54–62% germination. Radicles emerged 6 weeks after sowing and normal germination with emergence of the primary single shoot growth occurred after 12 weeks. There was no significant difference (P ≥ 0.05) between germination responses of seeds from Ghana and Cote d’Ivoire. Seeds did not germinate when dried to below ca. 30% MC. De-coating seeds or soaking intact seeds in a 70% ethanol solution increased germination to 82–92%, and also increased germination rate; however germination was not uniform. Because difficulties in germinating these seeds stem from the physical presence of the seed coats and the slow growth of the embryos contained therein, we recommend that fresh seeds from mature fruits be de-coated and immediately sown in preferably, an aerated medium (e.g., sawdust) for cultivation.     

Genetic analysis of falling number in three bi‐parental common winter wheat populations

The objective of the present study was to analyse the genetic basis of falling number in three winter wheat populations. Samples for falling number determination for each population originated from at least three test environments that were free from the occurrence of preharvest sprouting at harvest time. Quantitative trait locus (QTL) analysis employing falling number values from single environments identified eight, five and three QTL in the populations Dream/Lynx, Bussard/W332‐84 and BAUB469511/Format, respectively. A major QTL common to all three populations and consistently detected in each environment mapped to the long arm of chromosome 7B. The QTL was located to a similar genomic region as the previously described major QTL for high‐isoelectric point α‐amylase content. The T1BL.1RS wheat‐rye translocation and the dwarfing gene Rht‐D1 segregating in Dream/Lynx and BAUB469511/Format were found to be important factors of falling number variation. In both populations, the presence of Rht‐D1b or the absence of T1BL.1RS increased falling number. The results indicate that late maturity α‐amylase, responsible for low falling numbers, has now been documented in German wheat germplasm.     

Adhesion molecule and homing receptor expression on blood and milk polymorphonuclear leukocytes during the periparturient period of dairy cattle

Adhesion molecule and homing receptor expression on blood and milk polymorphonuclear leukocytes (PMN) from periparturient dairy cattle was studied. Both percentages and the mean fluorescence intensity (MFI) of PMN expressing CD11a, CD44, CD62L, and LPAM-1 (alpha4 beta7) were evaluated at seven time points during the twenty-one day period post calving. CD11a and CD62L were expressed on 94-100% of PMN in both blood and milk and there were no significant differences in these percentages at any time point. LPAM-1 was expressed on 3-10% of the PMN in the blood and 13-45% in the milk and the percentage of cells expressing LPAM-1 in milk was significantly (P<0.05) greater than in blood at 0, 4, 10, 14, 18 and 21 days after calving. CD44 was expressed on 11-39% of the PMN in blood and 33-69% in the milk and the percentage of cells expressing CD44 in milk was significantly (P<0.05) greater than in blood at all time points. The MFI of CD11a on milk PMN was consistently higher than that of blood PMN throughout the study period and significantly (P<0.05) higher at days 4, 10 and 18 after calving.     

Lymphocyte subsets and adhesion molecule expression in milk and blood of periparturient dairy cattle

Fifteen Holstein dairy cattle were monitored for lymphocyte subsets and expression of adhesion molecules on cells in milk and blood at parturition and at intervals up to 21 days post-partum. Using flow cytometry, we determined percentages of T cells (CD4+, CD8+, gammadelta) and B cells from milk and blood of these cows. We also measured expression of adhesion molecules (CD62L, LFA-1, LPAM-1, and CD44) on lymphocytes in milk and blood. Significantly higher percentages of CD8+ cells were found in milk than in blood at all time points while significantly higher percentages of B cells were found in blood than in milk at all time points. There were minimal to no significant differences in percentages of CD4+ or gammadelta+ cells between milk and blood. Expression of adhesion molecules was consistently higher on all subsets of milk lymphocytes compared with blood lymphocytes. These differences were most pronounced and statistically significant at calving and in the first week following calving. CD62L, LPAM-1 and CD44 were expressed on a significantly higher percentage of lymphocytes in milk at calving than in milk at subsequent sampling times, while LFA-1 expression on lymphocytes in milk was significantly lower at calving than at subsequent times.     

Surgical correction of subaortic stenosis via right ventriculotomy and septal resection in a dog

After 2 years of medical management with a beta-adrenoreceptor blocking agent, a 30-month-old castrated male Golden Retriever with subaortic stenosis was treated surgically because of progression of its condition. In an attempt to achieve complete relief of the left ventricular outflow obstruction, a modified Konno procedure consisting of right ventriculotomy and septal myectomy from the infundibular portion of the right ventricle was performed; this combination of procedures allowed wide resection of the septal portion of the left ventricular outflow obstruction. Two years after surgery, the mass of the dog's left ventricle had decreased and the peak calculated pressure gradient across the aortic valve had decreased to 40 mm Hg, compared with a preoperative value of 240 mm Hg; at that evaluation, the dog had gained weight and was able to play normally. It is suggested that use of this modified approach to the outflow tract may have a positive effect on long-term survival time in dogs with subaortic stenosis.