Recent advances in understanding cell types during human gastrulation.

Gastrulation is a fundamental process during embryonic development, conserved across all multicellular animals [1]. In the majority of metazoans, gastrulation is characterised by large scale morphogenetic remodeling, leading to the conversion of an early pluripotent embryonic cell layer into the three primary 'germ layers': an outer ectoderm, inner endoderm and intervening mesoderm layer. The morphogenesis of these three layers of cells is closely coordinated with cellular diversification, laying the foundation for the generation of the hundreds of distinct specialized cell types in the animal body. The process of gastrulation has for a long time attracted tremendous attention in a broad range of experimental systems ranging from sponges to mice. In humans the process of gastrulation starts approximately 14 days after fertilization and continues for slightly over a week. However our understanding of this important process, as it pertains to human, is limited. Donations of human fetal material at these early stages are exceptionally rare, making it nearly impossible to study human gastrulation directly. Therefore, our understanding of human gastrulation is predominantly derived from animal models such as the mouse [2,3] and from studies of limited collections of fixed whole samples and histological sections of human gastrulae [4-7], some of which date back to over a century ago. More recently we have been gaining valuable molecular insights into human gastrulation using in vitro models of hESCs [8-12] and increasingly, in vitro cultured human and non-human primate embryos [13-16]. However, while methods have been developed to culture human embryos into this stage (and probably beyond), current ethical standards prohibit the culture of human embryos past 14 days again limiting our ability to experimentally probe human gastrulation. This review discusses recent molecular insights from the study of a rare CS 7 human gastrula obtained as a live sample and raises several questions arising from this recent study that it will be interesting to address in the future using emerging models of human gastrulation.

Characterisation of infantile cardiomyopathy in Alström syndrome using ALMS1 knockout induced pluripotent stem cell derived cardiomyocyte model.

Alström syndrome (AS) is an inherited rare ciliopathy characterised by multi-organ dysfunction and premature cardiovascular disease. This may manifest as an infantile-onset dilated cardiomyopathy with significant associated mortality. An adult-onset restrictive cardiomyopathy may also feature later in life. Loss of function pathogenic variants in ALMS1 have been identified in AS patients, leading to a lack of ALMS1 protein. The biological role of ALMS1 is unknown, particularly in a cardiovascular context. To understand the role of ALMS1 in infantile cardiomyopathy, the reduction of ALMS1 protein seen in AS patients was modelled using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), in which ALMS1 was knocked out. MuscleMotion analysis and calcium optical mapping experiments suggest that ALMS1 knockout (KO) cells have increased contractility, with altered calcium extrusion and impaired calcium handling dynamics compared to wildtype (WT) counterparts. Seahorse metabolic assays showed ALMS1 knockout iPSC-CMs had increased glycolytic and mitochondrial respiration rates, with ALMS1 knockout cells portraying increased energetic demand and respiratory capacity than WT counterparts. Using senescence associated ß-galactosidase (SA-ß gal) staining assay, we identified increased senescence of ALMS1 knockout iPSC-CMs. Overall, this study provides insights into the molecular mechanisms in AS, particularly the role of ALMS1 in infantile cardiomyopathy in AS, using iPSC-CMs as a 'disease in a dish' model to provide insights into multiple aspects of this complex disease.

Biochemical characterisation and production kinetics of high molecular-weight (HMW) putative antibacterial proteins of insect pathogenic Brevibacillus laterosporus isolates.

BACKGROUND: Bacterial genomes often encode structures similar to phage capsids (encapsulins) and phage tails which can be induced spontaneously or using genotoxic compounds such as mitomycin C. These high molecular-weight (HMW) putative antibacterial proteins (ABPs) are used against the competitive strains under natural environment. Previously, it was unknown whether these HMW putative ABPs originating from the insect pathogenic Gram-positive, spore-forming bacterium Brevibacillus laterosporus (Bl) isolates (1821L, 1951) are spontaneously induced during the growth and pose a detrimental effect on their own survival. Furthermore, no prior work has been undertaken to determine their biochemical characteristics. RESULTS: Using a soft agar overlay method with polyethylene glycol precipitation, a narrow spectrum of bioactivity was found from the precipitated lysate of Bl 1951. Electron micrographs of mitomycin C- induced filtrates showed structures similar to phage capsids and contractile tails. Bioactivity assays of cell free supernatants (CFS) extracted during the growth of Bl 1821L and Bl 1951 suggested spontaneous induction of these HMW putative ABPs with an autocidal activity. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of spontaneously induced putative ABPs showed appearance of ~ 30 kDa and ~ 48 kDa bands of varying intensity across all the time intervals during the bacterial growth except in the initial hours. Statistically, spontaneously induced HMW putative ABPs of Bl 1951 exhibited a significant decrease in the number of viable cells of its producer strain after 18 h of growth in liquid. In addition, a significant change in pH and prominent bioactivity of the CFS of this particular time period was noted. Biochemically, the filtered supernatant derived from either Bl 1821L or Bl 1951 maintained bioactivity over a wide range of pH and temperature. CONCLUSION: This study reports the spontaneous induction of HMW putative ABPs (bacteriocins) of Bl 1821L and Bl 1951 isolates during the course of growth with potential autocidal activity which is critically important during production as a potential biopesticide. A narrow spectrum of putative antibacterial activity of Bl 1951 precipitate was found. The stability of HMW putative ABPs of Bl 1821L and Bl 1951 over a wide range of pH and temperature can be useful in expanding the potential of this useful bacterium beyond the insecticidal value.

Application of various microscopy techniques to study early-age and longer-term behaviour of super sulphated cement microstructure.

Super sulphated cement (SSC) is a very promising substitute for traditional construction materials (i.e. Portland cement), due to its enhanced durability and particularly low environmental impact. This paper explores the microstructure and certain properties of SSC, focusing on the particular complexities of its microstructure and the difficulties of microanalysis of its hydrates. To do so, SSC paste samples were first cast to identify hydration products using X-ray diffraction, then observed at early age using confocal laser scanning microscopy (CLSM) and at early and late age using scanning electron microscopy. In addition, concrete cores impregnated with fluorescein in order to highlight porosity, cracking and aggregates debonding were observed under UV light using optical microscopy (OM), showing a complete absence of cracking and aggregate debonding. Both microscopy techniques (CLSM and UV light OM) have been applied to this type of binder for the first time. The results show that SSC microstructure is characterised by a sophisticated intergrowth of various phases, including ettringite and amorphous calcium-(alumina)-silicate hydrate gels. Finally, Monte-Carlo simulation of electron-matter has been provided for a better understanding of EDS analysis.

Clinker based on calcium looped meals from the Cleanker Project.

The Cleanker (CLEAN clinKER production by Calcium Looping process) project, financed in the framework of Horizon 2020 EU funding program, has demonstrated the feasibility of the integrated CaL concept at industrial scale in a new demo system realised in the Buzzi Unicem cement plant in Vernasca (IT). The Calcium Looping (CaL) CO2 capture process exploits the reversible reaction of limestone calcination/carbonation (CaCO3 ↔ CaO + CO2). The Cleanker pilot plant consists of the coupling of a carbonator and an oxyfuel calciner. When the flue gases from kiln (containing CO2) flow through the carbonator together with calcined meal, which acts as a CO2 sorbent, carbonation takes place and CO2 is fixed in calcium carbonate. CaO is then regenerated in the calciner, where the opposite reaction takes place, and the captured CO2 is released. When the lime/limestone looping is carried out through a carbonator and a calciner operated in oxyfuel, a concentrated CO2 stream is obtained that can be efficiently addressed towards CCS or CCUS processes. In the Cleanker Project, the possibility to use raw meal for clinker production has been exploited and the meals sampled at the outlet of the oxyfuel calciner have been characterised and used for producing clinker at a lab scale. Calcium looped (CaL) meals collected at different sampling point during the operation of the CaL plant were characterised by means of X-ray fluorescence spectroscopy, X-ray diffraction and particle size distribution. Electron scanning microscopy revealed significant differences in the surface of particles of the looped meals with respect to the original one, resulting from the loading and unloading of CO2 during the looping cycles. The meals were burnt in a laboratory furnace and the obtained clinkers have been characterised on a chemical, mineralogical and microscopical point of view, revealing the good burnability of all the meals and attesting the possibility to reintegrate the materials in the clinker production process. A lower alite/belite ratio in the clinker produced from the looped meals was observed: actually, the depletion of calcium during the recirculation of the meal in the calciner/carbonator system led to a reduction of the lime saturation factor influencing the mineralogical composition of the product.

Impact of different peak tube voltage settings on adrenal adenomas attenuation at unenhanced CT.

OBJECTIVES: To assess the influence of peak tube voltage peak setting on adrenal adenomas (AA) attenuation on unenhanced abdominal CT. MATERIALS AND METHODS: IRB-approved retrospective observational cohort study. We included 89 patients with imaging-defined AAs with shortest diameter > 6 mm who underwent two or more unenhanced abdominal CTs using at least two different peak tube voltage settings. Two readers independently measured adenoma attenuation on different CT acquisitions by drawing a round ROI on 3 mm thick axial MPR reconstructions encompassing at least 2/3 of the lesion's surface. The mean of the values measured by the two readers was used for further analysis. Interobserver variability was assessed (Intraclass Correlation Coefficient). Attenuation values measured on 100, 110 and 140 kVp acquisitions were compared with standard 120 kVp ones (Bland-Altman analysis). RESULTS: We included 275 unenhanced abdominal CTs (3.1 ± 0.9/patient) in image analysis; 131 acquired at 120 kVp, 65 at 100 kVp, 59 at 110 kVp, and 20 at 140 kVp. 107 lesions were detected in 89 patients (1-4/patient), with a mean maximum diameter of 17 ± 6 mm. Interobserver agreement in attenuation measurement was excellent (ICC: 0.95, CI (92-97)). Median adenoma attenuation was significantly lower on 100 kVp images than on 120 kVp ones (-1 HU, IQR (-5 to 3.6), vs, 2.5 HU, IQR (-1.5 to 8.5); p < 0.001) whereas we didn't find statistically significant differences in adenoma attenuation between 110 kVp or 140 kVp and 120 kVp ones. CONCLUSION: AA attenuation is significantly lower on unenhanced CT scans acquired at 100 kVp than on those acquired at "standard" 120 kVp. CLINICAL RELEVANCE STATEMENT: AA attenuation is significantly lower at 100 kVp in comparison to 120 kVp. This might be exploited to increase unenhanced CT sensitivity in adenoma characterisation, but further studies including non-adenoma lesions are mandatory to confirm this hypothesis. KEY POINTS: CT scans are often acquired using peak tube voltage settings different from the "standard" 120 kVp. AA attenuation varies if CT scans are acquired using different tube peak voltage settings. At 100 kVp AAs show a significantly lower attenuation than at 120 kVp.

Cold Storage of Human Femoral Arteries for Twelve Months: Impact on Mechanical Properties.

OBJECTIVE: This biomechanical pre-clinical study aimed to assess the consequences on mechanical properties of long term cold storage (+2 to +8 °C) of arterial allografts. METHODS: Femoropopliteal arterial segments were collected from multiorgan donors and stored at +2 to +8 °C for twelve months in saline solution with added antibiotics. Mechanical characterisation was carried out using two different tests, with the aim of defining the physiological modulus and the maximum stress and strain borne by the sample before rupture. These characterisations were carried out after zero, six, and twelve months of storage for each sample (T0, T6, and T12, respectively). For comparison, the same tests were performed on cryopreserved femoropopliteal segments after thawing. RESULTS: Twelve refrigerated allografts (RAs), each divided into three segments, and 10 cryopreserved allografts (CAs) were characterised. The median (interquartile range [IQR]) Young's modulus was not statistically significantly different between the storage times for cold stored allografts: RAT0, 164 (150, 188) kPa; RAT6, 178 (141, 185) kPa; RAT12, 177 (149, 185) kPa. The median (IQR) Young's modulus of the CA group (153; 130, 170 kPa) showed no significant differences from the RA groups, irrespective of storage time. Furthermore, median (IQR) maximum stress and strain values were not significantly different between the different groups: for maximum stress: RAT0, 1.58 (1.08, 2.09) MPa; RAT6, 1.74 (1.55, 2.36) MPa; RAT12, 2.25 (1.87, 2.53) MPa; CA, 2.25 (1.77, 2.61) MPa; and for maximum strain: RAT0, 64% (50, 90); RAT6, 79% (63, 84); RAT12, 72% (65, 86); CA, 67% (50, 95). CONCLUSION: Cold storage for up to twelve months appears to have no impact on the mechanical characteristics of human arterial allografts. Therefore, this preservation method, which would greatly simplify routine care, seems feasible. Other indicators are being studied to verify the safety of this preservation process before considering its use in vivo.

Virulence evaluation of Israeli Marek's disease virus isolates from commercial poultry using their meq gene sequence.

Fifty-seven Gallid alphaherpesvirus 2 (GaHV-2) isolates, collected during a 30-year period (1990-2019) from commercial poultry flocks affected by Marek's disease (MD), were molecularly characterised. The GaHV-2 meq gene was amplified and sequenced to evaluate the virus virulence, based on the number of PPPPs within the proline-rich repeats (PRRs) of its transactivation domain. The present illustration of virus virulence evaluation on a large scale of field virus isolates by molecular analysis exemplifies the practical benefit and usefulness of the molecular marker in commercial GaVH-2 isolates. The alternative assay of GaVH-2 virulence pathotyping is the classical Gold Standard ADOL method, which is difficult and impossible to employ on a large scale using the Specific Pathogen Free (SPF) chicks of the ADOL strains kept in isolators for two months. The phylogenetic analysis performed in the present study showed that the meq gene amino acid sequences of the 57 Israeli strains divide into 16 phylogenetic branches. The virulence evaluation was performed in comparison with 36 GaHV-2 prototype strains, previously characterised by the in vivo Gold Standard ADOL assay. The results obtained revealed that the GaHV-2 strains circulating in Israel have evolved into a higher virulence potential during the years, as the four-proline stretches number in the meq gene decreased over the investigated period, typically of very virulent virus prototypes. The present study supports the meq gene molecular markers for the assessment of field GaVH-2 strains virulence.

Optimization of hydrochar synthesis conditions for enhanced Cd(II) and Pb(II) adsorption in mono and multimetallic systems.

The characterisation of hydrochars derived from Sargassum biomass collected along the Mexican Caribbean coast reveals their favourable morphology and chemical composition for incorporating metal ions, including Cd(II) and Pb(II). Among the synthesized materials, HCS-3, produced at 180 °C with a 2 h residence time, exhibited superior yield, specific area, carbon content, and capacity for removing Cd(II) and Pb(II). Adsorption equilibrium studies demonstrate the presence of adsorption processes during Cd(II) and Pb(II) retention on HCS-3, with adsorption capacities slightly exceeding 140 and 340 mg g⁻1, respectively. Notably, HCS-3 shows a greater affinity for Pb(II) over Cd(II) when both elements are present concurrently. The physicochemical analysis through FTIR spectroscopy, functional group analysis, point of zero charge determination, SEM/EDS, and other techniques evidenced that HCS-3 possesses favourable characteristics to serve as a heavy metal adsorbent. These findings underscore the efficacy of hydrochars from Sargassum biomass in removing heavy metals, suggesting their potential as superior adsorbents compared to traditional or novel materials, and advising its possible versatility for other pollutants. Utilizing these hydrochars could mitigate the economic and environmental impact of Sargassum biomass by repurposing it for valuable applications.

Co-circulation of seasonal influenza A(H1N1)pdm09, A(H3N2) and B/Victoria lineage viruses with further genetic diversification, EU/EEA, 2022/23 influenza season.

BackgroundInfluenza viruses can cause large seasonal epidemics with high healthcare impact and severity as they continually change their virological properties such as genetic makeup over time.AimWe aimed to monitor the characteristics of circulating influenza viruses over the 2022/23 influenza season in the EU/EEA countries. In addition, we wanted to compare how closely the circulating viruses resemble the viral components selected for seasonal influenza vaccines, and whether the circulating viruses had acquired resistance to commonly used antiviral drugs.MethodsWe performed a descriptive analysis of the influenza virus detections and characterisations reported by National Influenza Centres (NIC) from the 30 EU/EEA countries from week 40/2022 to week 39/2023 to The European Surveillance System (TESSy) as part of the Global Influenza Surveillance and Response System (GISRS).ResultsIn the EU/EEA countries, the 2022/23 influenza season was characterised by co-circulation of A(H1N1)pdm09, A(H3N2) and B/Victoria-lineage viruses. The genetic evolution of these viruses continued and clade 6B.1A.5a.2a of A(H1N1)pdm09, 3C.2a1b.2a.2b of A(H3N2) and V1A.3a.2 of B/Victoria viruses dominated. Influenza B/Yamagata-lineage viruses were not reported.DiscussionThe World Health Organization (WHO) vaccine composition recommendation for the northern hemisphere 2023/24 season reflects the European virus evolution, with a change of the A(H1N1)pdm09 component, while keeping the A(H3N2) and B/Victoria-lineage components unchanged.