Identification of ARUK2002821 as an isoform-selective PI5P4Kα inhibitor.

The phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) play a central role in regulating cell signalling pathways and, as such, have become therapeutic targets for diseases such as cancer, neurodegeneration and immunological disorders. Many of the PI5P4Kα inhibitors that have been reported to date have suffered from poor selectivity and/or potency and the availability of better tool molecules would facilitate biological exploration. Herein we report a novel PI5P4Kα inhibitor chemotype that was identified through virtual screening. The series was optimised to deliver ARUK2002821 (36), a potent PI5P4Kα inhibitor (pIC50 = 8.0) which is selective vs. other PI5P4K isoforms and has broad selectivity against lipid and protein kinases. ADMET and target engagement data are provided for this tool molecule and others in the series, as well as an X-ray structure of 36 solved in complex with its PI5P4Kα target.

[The experience of community pharmacy in SARS-CoV-2 antigen detection tests programs.] / Experiencia de la farmacia comunitaria en los programas de pruebas de detección de antígenos del SARS-CoV-2.

SARS-CoV-2 infection was an unprecedented pandemic with unprecedented global health and socio-economic impact. More than 13 million cases had been confirmed in Spain by August 2022, and diagnostic testing to detect cases of infection in the country has helped to partially mitigate the spread of the virus. In 2021, the first self-testing antigen tests were marketed for dispensing in community pharmacies, and over-the-counter dispensing was allowed from July of that year. The network of community pharmacies played a key role, not only in the informed dispensing of these tests, but also in actively participating in the performance, supervision and reporting of results to the health authorities, and even in the issuing of digital certificates. A compilation has been made of all the available data on the subject, with a deadline of 13 February 2022, which is considered to be the end of the sixth wave of the epidemic in Spain. The results of the action taken by community pharmacies in twelve Autonomous Communities, which somehow participated in these initiatives by carrying out or supervising a total of 1,043,800 tests, from which 109,570 positive cases (10.5% of the total) were detected and reported to the National Health System, are presented in this article. Although the results are provisional, because many of the programmes are still ongoing, they are a clear demonstration of the potential that community pharmacies can play in Public Health work.

La infección por SARS-CoV-2 ha constituido una pandemia con un impacto sanitario y socioeconómico global sin precedentes. Con más de trece millones de casos confirmados en España hasta agosto de 2022, la realización de pruebas diagnósticas para detectar los casos de infección ha permitido atenuar parcialmente la expansión del virus. Durante 2021 se comercializaron los primeros test de antígenos para autodiagnóstico, de dispensación en farmacias comunitarias, y desde julio de ese año se permitió su dispensación sin receta médica. La red de farmacias comunitarias jugó un papel fundamental, no solo por la dispensación informada de dichos test, sino participando activamente en la realización, en la supervisión de su realización y en la notificación de resultados a las autoridades sanitarias, e incluso en la emisión de certificados digitales. Se ha realizado una recopilación de todos los datos disponibles al respecto, fijando como límite temporal la semana del 13 de febrero de 2022, por considerarse como el final de la sexta ola de la epidemia en España. El presente artículo revela los resultados derivados de la actuación de las farmacias de doce comunidades autónomas, que participaron de una forma u otra en dichas iniciativas mediante la realización o supervisión de un total de 1.043.800 pruebas, a partir de las cuales se detectaron 109.570 casos positivos (un 10,5% del total), que fueron comunicados al Sistema Nacional de Salud. Los resultados son provisionales, pues muchos de los programas continúan vigentes, pero son una muestra inequívoca del potencial que las farmacias comunitarias pueden desempeñar en tareas de Salud Pública.

Postbiotic yeast fermentation product supplementation to lactating goats increases the efficiency of milk production by enhancing fiber digestibility and ruminal propionate, and reduces energy losses in methane.

Although in vitro data with mixed ruminal fluid demonstrated positive effects of posbiotic diet (POS) from lactobacilli on measures of fermentation and microbial profiles, there is a paucity of in vivo data with lactating ruminants. The aim of the study was to evaluate the effects of incorporating POS into diets of lactating goats on energy (E) partitioning, carbon (C) and nitrogen (N) balance, and performance. Ten late-lactation Murciano-Granadina goats were used in a crossover design with 26-d periods. Goats in the control diet (CON) were fed daily at the rate of 1 kg alfalfa hay and 1.5 kg concentrate, and the treatment group (POS) was fed CON with the addition of 3.75 g/d of Probisan Ruminants (PENTABIOL S.L., Navarra, Spain). No differences in DMI were detected. However, ruminal fluid propionate and apparent total tract digestibilities of NDF and ADF were greater (18%, 4.7%, and 5.2%, respectively; P < 0.05) in POS compared with the CON diet. Daily partitioning of E to milk and efficiency of ME intake for milk production greater (11% and 3.0%, respectively; P < 0.05) in POS compared with CON. The nonprotein RQ was greater in POS compared with CON due to greater (P < 0.05) oxidation of carbohydrate (213 vs. 115 kJ/kg of BW0.75 per day) compared with fat (362 vs. 486 kJ/kg of BW0.75 per day). Although no differences were found in C balance, goats in POS had lower (P < 0.05) amounts of C in CH4 (1.1 vs. 1.3 g/kg BW0.75 per day) compared with CON. There were no differences in N intake or N in feces or urine, but N in milk was greater (P < 0.05) in POS compared with the CON diet (0.8 vs. 0.7 g/kg BW0.75 per day). Yield of fat-corrected milk (FCM) (3.20 vs. 2.72 kg/d; P < 0.05) and concentration of true protein (3.4 vs. 3.3 kg/d; P < 0.05) and lactose (4.7 vs. 4.5 kg/d; P < 0.05) were greater in POS compared with CON. These responses were accompanied by lower (P < 0.05) urea (12.3 vs. 16.6 mM/L) and ammonia-N (6.6 vs. 8.8 mg/L) without changes in fat concentration (6.1% vs. 6.0%; P > 0.05) in POS compared with the CON diet. Daily amount of CH4 emission did not differ P > 0.05 between diets. However, when expressed relative to unit of edible product, feeding POS reduced (P < 0.05) the amount of CH4 by 46 g/kg of milk fat, 97 g/kg of milk protein, and 3 g/kg of milk compared with CON. Overall, data indicated that feeding a postbiotic in late-lactation increased energy efficiency for milk production partly by reducing CH4 emission.

Although in vitro data with mixed ruminal fluid demonstrated positive effects of postbiotics from lactobacilli on measures of fermentation and microbial profiles, there is a paucity of in vivo data with lactating ruminants. We evaluated the effects of incorporating a postbiotic yeast fermentation product in diets of lactating goats on energy partitioning, carbon and nitrogen balance, and performance. The postbiotic led to greater ruminal propionate concentration and fiber digestibility, and decreased partitioning of energy to methane. Those changes were associated with greater milk production. Data suggested that postbiotics could enhance efficiency of nutrient use for milk production.

Development of Selective Phosphatidylinositol 5-Phosphate 4-Kinase γ Inhibitors with a Non-ATP-competitive, Allosteric Binding Mode.

Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are emerging as attractive therapeutic targets in diseases, such as cancer, immunological disorders, and neurodegeneration, owing to their central role in regulating cell signaling pathways that are either dysfunctional or can be modulated to promote cell survival. Different modes of binding may enhance inhibitor selectivity and reduce off-target effects in cells. Here, we describe efforts to improve the physicochemical properties of the selective PI5P4Kγ inhibitor, NIH-12848 (1). These improvements enabled the demonstration that this chemotype engages PI5P4Kγ in intact cells and that compounds from this series do not inhibit PI5P4Kα or PI5P4Kß. Furthermore, the first X-ray structure of PI5P4Kγ bound to an inhibitor has been determined with this chemotype, confirming an allosteric binding mode. An exemplar from this chemical series adopted two distinct modes of inhibition, including through binding to a putative lipid interaction site which is 18 Å from the ATP pocket.

Alterations in Energy Partitioning and Methane Emissions in Murciano-Granadina Goats Fed Orange Leaves and Rice Straw as a Replacement for Beet Pulp and Barley Straw.

Considering the huge quantities of crops by-products and pruning waste such as rice straw and citrus leaves produced annually worldwide, and their potential pollution capacity, recycling as feed for livestock is an alternative. The objective was to study these by-products effect on energy balance and methane emissions in 10 Murciano-Granadina goats at maintenance. The control diet (CTR) included barley straw and beet pulp while the experimental diet (ORG) consisted of rice straw and orange leaves. Differences were found for energy intake (248 kJ/kg of BW0.75 greater for CTR than ORG). The intake of metabolizable energy was 199 kJ/kg of BW0.75 lower in ORG than CTR, and the energy efficiency was higher with CTR (0.61) than ORG (0.48). Protein retained in the body was 9 g/goat greater with CTR than ORG, and fat retention in the body was approximately 108 g/goat greater with CTR than ORG. Despite more unfavorable energy balance in response to feeding ORG than CTR, the retention of body energy was always positive. Reductions in CH4 emissions were detected when goats were fed ORG diet (from 22.3 to 20.0 g/d). Overall results suggested that feeding orange leaves and rice straw was effective in reducing CH4 emissions without adversely affecting energy balance.

Hoxa9 and EGFP reporter expression in human Embryonic Stem Cells (hESC) as useful tools for studying human development.

HoxA9 is an evolutionarily conserved homeobox gene implicated in embryo development. To study the roles of Hoxa9 during human development we generated a transgenic H9 (hESC) line that overexpresses HoxA9 and the Enhanced Green Fluorescent Protein (EGFP), and a control H9 with a stable expression of the EGFP. The resulting H9-HoxA9-EGFP and H9-EGFP cell lines allow an efficient visualization of hESCs by fluorescent microscopy, quantification by flow cytometry and cell differentiation tracking. Both transgenic cell lines maintained the pluripotent phenotype, the ability to differentiate into all three germ layers and a normal karyotype.

RUNX1c Regulates Hematopoietic Differentiation of Human Pluripotent Stem Cells Possibly in Cooperation with Proinflammatory Signaling.

Runt-related transcription factor 1 (Runx1) is a master hematopoietic transcription factor essential for hematopoietic stem cell (HSC) emergence. Runx1-deficient mice die during early embryogenesis due to the inability to establish definitive hematopoiesis. Here, we have used human pluripotent stem cells (hPSCs) as model to study the role of RUNX1 in human embryonic hematopoiesis. Although the three RUNX1 isoforms a, b, and c were induced in CD45+ hematopoietic cells, RUNX1c was the only isoform induced in hematoendothelial progenitors (HEPs)/hemogenic endothelium. Constitutive expression of RUNX1c in human embryonic stem cells enhanced the appearance of HEPs, including hemogenic (CD43+) HEPs and promoted subsequent differentiation into blood cells. Conversely, specific deletion of RUNX1c dramatically reduced the generation of hematopoietic cells from HEPs, indicating that RUNX1c is a master regulator of human hematopoietic development. Gene expression profiling of HEPs revealed a RUNX1c-induced proinflammatory molecular signature, supporting previous studies demonstrating proinflammatory signaling as a regulator of HSC emergence. Collectively, RUNX1c orchestrates hematopoietic specification of hPSCs, possibly in cooperation with proinflammatory signaling. Stem Cells 2017;35:2253-2266.

Albendazole residues in goat's milk: Interferences in microbial inhibitor tests used to detect antibiotics in milk.

Albendazole (ABZ) residues in goat's milk and their effect on the response of microbial inhibitor tests used for screening antibiotics were evaluated. A total of 18 Murciano-Granadina goats were treated with ABZ and individually milked once a day over a 7-day period. ABZ quantification was performed by high performance liquid chromatography. The ABZ parent drug was not detected. The maximum concentration of its metabolites (ABZ sulfoxide, ABZ sulfone, and ABZ 2-aminosulfone) was reached on the 1st day post treatment (260.0 ± 70.1 µg/kg, 112.8 ± 28.7 µg/kg, 152.0 ± 23.6 µg/kg, respectively), decreasing to lower than the maximum residue limit (MRL, 100 µg/kg) on the 3rd day post treatment. Milk samples were also analyzed by microbial tests [Brilliant Black Reduction Test (BRT) MRL, Delvotest SP-NT MCS and Eclipse 100], and only one positive result was found for Delvotest SP-NT MCS and Eclipse 100. However, a high occurrence of positive outcomes was obtained for BRT MRL during 6 days post treatment, whereas ABZ residues were not detected from the 4th day post administration, suggesting that factors other than the antiparasitic agent might affect the microbial test response.

Interferences on microbial inhibitor tests related to ivermectin treatment in lactating dairy goats.

This Research Communication reports interferences related to the administration of ivermectin in lactating dairy goats on the response of microbial tests for screening antibiotics in milk. Twenty-eight Murciano-Granadina goats, naturally infested with Sarcoptes scabiei var. caprae, were treated with a subcutaneous injection of ivermectin (200 µg/kg b.w.). To prevent re-infestation, a second dose was applied 7 d later. Individual milk samples were collected, daily, up to 15 d post-treatment. Milk samples were analysed by microbial inhibitor tests (BRT MRL, Delvotest SP-NT MCS and Eclipse 100) and ivermectin residues were quantified by HPLC. A large number of positive results were obtained for all microbial tests, especially on the first day after treatment (BRT MRL = 46·4%; Delvotest SP-NT MCS = 14·3%; and Eclipse 100 = 17·8%). However, the highest concentration of drug residues in milk (24·3 ng/ml) was detected on the tenth day after treatment, when positive outcomes were relatively lower (BRT MRL = 17·8%; Delvotest SP-NT MCS = 10·7%; and Eclipse 100 = 7·4%). Results herein suggest that factors related to the ivermectin treatment other than drug residues in milk, or alterations produced by the parasitic disease itself affecting the immune response of animals, could be the cause of false-positive results in microbial tests. It can be concluded that the application of ivermectin in dairy goats infested with sarcoptes mange during lactation produces persistent drug residues in milk, and could also cause false-positive results in microbial inhibitor tests for screening antibiotics.

Generation of induced pluripotent stem cells (iPSCs) from a Bernard-Soulier syndrome patient carrying a W71R mutation in the GPIX gene.

We generated an induced pluripotent stem cell (iPSC) line from a Bernard-Soulier Syndrome (BSS) patient carrying the mutation p.Trp71Arg in the GPIX locus (BSS1-PBMC-iPS4F4). Peripheral blood mononuclear cells (PBMCs) were reprogrammed using heat sensitive non-integrative Sendai viruses containing the reprogramming factors Oct3/4, SOX2, KLF4 and c-MYC. Successful silencing of the exogenous reprogramming factors was checked by RT-PCR. Characterization of BSS1-PBMC-iPS4F4 included mutation analysis of GPIX locus, Short Tandem Repeats (STR) profiling, alkaline phosphatase enzymatic activity, analysis of conventional pluripotency-associated factors at mRNA and protein level and in vivo differentiation studies. BSS1-PBMC-iPS4F4 will provide a powerful tool to study BSS.

Generation of human iPSC line GRX-MCiPS4F-A2 from adult peripheral blood mononuclear cells (PBMCs) with Spanish genetic background.

We have generated iPSCs from peripheral blood mononuclear cells (PBMCs) of a healthy man using heat sensitive and non-integrative Sendai virus containing Sox2, Oct3/4, c-Myc and Klf4. Human GRX-MCiPS4F-A2 cell line was established and characterized through this study.

Generation and characterization of the human iPSC line PBMC1-iPS4F1 from adult peripheral blood mononuclear cells.

Here we describe the generation and characterization of the human induced pluripotent stem cell (iPSC) line PBMC1-iPS4F1 from peripheral blood mononuclear cells from a healthy female with Spanish background. We used heat sensitive, non-integrative Sendai viruses containing the reprogramming factors Oct3/4, Sox2, Klf4 and c-Myc, whose expression was silenced in the established iPSC line. Characterization of the PBMC1-iPS4F1 cell line included analysis of typical pluripotency-associated factors at mRNA and protein level, alkaline phosphatase enzymatic activity, and in vivo and in vitro differentiation studies.

SCL/TAL1-mediated transcriptional network enhances megakaryocytic specification of human embryonic stem cells.

Human embryonic stem cells (hESCs) are a unique in vitro model for studying human developmental biology and represent a potential source for cell replacement strategies. Platelets can be generated from cord blood progenitors and hESCs; however, the molecular mechanisms and determinants controlling the in vitro megakaryocytic specification of hESCs remain elusive. We have recently shown that stem cell leukemia (SCL) overexpression accelerates the emergence of hemato-endothelial progenitors from hESCs and promotes their subsequent differentiation into blood cells with higher clonogenic potential. Given that SCL participates in megakaryocytic commitment, we hypothesized that it may potentiate megakaryopoiesis from hESCs. We show that ectopic SCL expression enhances the emergence of megakaryocytic precursors, mature megakaryocytes (MKs), and platelets in vitro. SCL-overexpressing MKs and platelets respond to different activating stimuli similar to their control counterparts. Gene expression profiling of megakaryocytic precursors shows that SCL overexpression renders a megakaryopoietic molecular signature. Connectivity Map analysis reveals that trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), both histone deacetylase (HDAC) inhibitors, functionally mimic SCL-induced effects. Finally, we confirm that both TSA and SAHA treatment promote the emergence of CD34(+) progenitors, whereas valproic acid, another HDAC inhibitor, potentiates MK and platelet production. We demonstrate that SCL and HDAC inhibitors are megakaryopoiesis regulators in hESCs.

HOXA9 promotes hematopoietic commitment of human embryonic stem cells.

The molecular determinants regulating the specification of human embryonic stem cells (hESCs) into hematopoietic cells remain elusive. HOXA9 plays a relevant role in leukemogenesis and hematopoiesis. It is highly expressed in hematopoietic stem and progenitor cells (HSPCs) and is downregulated upon differentiation. Hoxa9-deficient mice display impaired hematopoietic development, and deregulation of HOXA9 expression is frequently associated with acute leukemia. Analysis of the genes differentially expressed in cord blood HSPCs vs hESC-derived HSPCs identified HOXA9 as the most downregulated gene in hESC-derived HSPCs, suggesting that expression levels of HOXA9 may be crucial for hematopoietic differentiation of hESC. Here we show that during hematopoietic differentiation of hESCs, HOXA9 expression parallels hematopoietic development, but is restricted to the hemogenic precursors (HEP) (CD31(+)CD34(+)CD45(-)), and diminishes as HEPs differentiate into blood cells (CD45(+)). Different gain-of-function and loss-of-function studies reveal that HOXA9 enhances hematopoietic differentiation of hESCs by specifically promoting the commitment of HEPs into primitive and total CD45(+) blood cells. Gene expression analysis suggests that nuclear factor-κB signaling could be collaborating with HOXA9 to increase hematopoietic commitment. However, HOXA9 on its own is not sufficient to confer in vivo long-term engraftment potential to hESC-hematopoietic derivatives, reinforcing the idea that additional molecular regulators are needed for the generation of definitive in vivo functional HSPCs from hESC.

Detection of antibiotics in goat's milk: effect of detergents on the response of microbial inhibitor tests.

The aim of the study was to evaluate the interference of acid and alkaline detergents employed in the cleaning of milking equipment of caprine dairy farms on the performance of microbial tests used in antibiotic control (BRT MRL, Delvotest MCS, and Eclipse 100). Eight concentrations of commercial detergents, five acid (0-0.25%) and five alkaline (0-1%) were add to antimicrobial-free goat's milk to evaluate the detergent effect on the response of microbial inhibitor tests. To evaluate the effect of detergents on the detection capability of microbial tests two detergents at 0.5 ml/l (one acid and one basic) and eight concentrations of four ß-lactam antibiotics (ampicillin, amoxicillin, cloxacillin and benzylpenicillin) were used. Milk without detergents was used as control. The spiked samples were analysed twelve times by three microbial tests. The results showed that the presence of acid detergents did not affect the response of microbial tests for any of the concentrations tested. However, at concentrations equal to or greater than 2 ml/l alkaline detergents positive results were found in microbial tests (16.7-100%). The detection limits of the screening tests for penicillins were not modified substantially by the presence of detergents. In general, the presence of acid and alkaline detergents in goat's milk did not produce a great interference in the microbial tests, only high concentrations of detergents could cause non-compliant results, but these concentrations are difficult to find in practice if proper cleaning procedures are applied in goat dairy farms.