Major Site Research Articles

An updated strong-motion database of Türkiye (SMD-TR)

We updated the earthquake strong-motion database of Türkiye (SMD-TR) that is initially compiled by Akkar et al. The updated data set consists of of 9244 earthquakes of magnitudes 3.0 ≤ M ≤ 7.8 that occurred between 1976 and 2023. It includes 95,890 three-component waveforms recorded at 1022 stations (55,375 records that are processed from 6710 events and 974 stations). The database is disseminated under research-oriented website DesignSafe ( https://doi.org/10.17603/ds2-f21x-s189 ) and is compiled to provide ground-motion flatfile, which will be updated periodically until 2025. The released version includes events occurred in Türkiye until 28 February 2023, and hence, it comprises of the mainshocks and a great portion of aftershocks from the February 6th, 2023, Kahramanmaraş earthquakes. The database includes event metadata compiled from national and international seismological agencies as well as the literature. We classified events according to their tectonic environments (i.e. active crustal and subduction) and identified the aftershocks. The station information provided by AFAD1 was used to compute the major site parameters, including time-based average of shear-wave velocity of uppermost 30 m (VS30) and depth-to-rock horizon at which the shear-wave velocity (VS) attains 1 km/s (Z1). We also computed the finite-fault distance metrics and flagged the stations located on the hanging-wall side of the rupture, as well as identified pulse-like records. We developed an automatic processing algorithm to determine the waveform quality, apply appropriate filtercut-offs to remove low- and high-frequency noise, and compute ground-motion parameters. The automatic processing was applied to small-magnitude (M < 5.5) events, whereas manual processing scheme is preferred for waveforms recorded from larger events (M ≥ 5.5). The peak ground-motion values, spectral ordinates, and ground-motion duration are provided along with the key data processing parameters in the flatfile.

Structural basis of nuclear transport for NEIL DNA glycosylases mediated by importin-alpha

NEIL glycosylases, including NEIL1, NEIL2, and NEIL3, play a crucial role in the base excision DNA repair pathway (BER). The classical importin pathway mediated by importin α/β and cargo proteins containing nuclear localization sequences (NLS) is the most common transport mechanism of DNA repair proteins to the nucleus. Previous studies have identified putative NLSs located at the C-terminus of NEIL3 and NEIL1. Crystallographic, bioinformatics, calorimetric (ITC), and fluorescence assays were used to investigate the interaction between NEIL1 and NEIL3 putative NLSs and importin-α (Impα). Our findings showed that NEIL3 contains a typical cNLS, with medium affinity for the major binding site of Impα. In contrast, crystallographic analysis of NEIL1 NLS revealed its binding to Impα, but with high B-factors and a lack of electron density at the linker region. ITC and fluorescence assays indicated no detectable affinity between NEIL1 NLS and Impα. These data suggest that NEIL1 NLS is a non-classical NLS with low affinity to Impα. Additionally, we compared the binding mode of NEIL3 and NEIL1 with Mus musculus Impα to human isoforms HsImpα1 and HsImpα3, which revealed interesting binding differences for HsImpα3 variant. NEIL3 is a classical medium affinity monopartite NLS, while NEIL1 is likely to be an unclassical low-affinity bipartite NLS. The base excision repair pathway is one of the primary systems involved in repairing DNA. Thus, understanding the mechanisms of nuclear transport of NEIL proteins is crucial for comprehending the role of these proteins in DNA repair and disease development.

Modelling and Molecular Dynamics Predict the Structure and Interactions of the Glycine Receptor Intracellular Domain.

Glycine receptors (GlyRs) are glycine-gated inhibitory pentameric ligand-gated ion channels composed of α or α + β subunits. A number of structures of these proteins have been reported, but to date, these have only revealed details of the extracellular and transmembrane domains, with the intracellular domain (ICD) remaining uncharacterised due to its high flexibility. The ICD is a region that can modulate function in addition to being critical for receptor localisation and clustering via proteins such as gephyrin. Here, we use modelling and molecular dynamics (MD) to reveal details of the ICDs of both homomeric and heteromeric GlyR. At their N and C ends, both the α and β subunit ICDs have short helices, which are major sites of stabilising interactions; there is a large flexible loop between them capable of forming transient secondary structures. The α subunit can affect the β subunit ICD structure, which is more flexible in a 4α2:1β than in a 4α1:1β GlyR. We also explore the effects of gephyrin binding by creating GlyR models bound to the gephyrin E domain; MD simulations suggest these are more stable than the unbound forms, and again there are α subunit-dependent differences, despite the fact the gephyrin binds to the β subunit. The bound models also suggest that gephyrin causes compaction of the ICD. Overall, the data expand our knowledge of this important receptor protein and in particular clarify features of the underexplored ICD.

Spatial Environment and Open Defecation: In Pursuit of Social Valuation of Sanitation Ecosystem Services

This article examines the social valuation of the external environment used for open defecation (OD). For this, we distinguish between the necessity-driven OD hypothesis that emanates from the reasoning of scarcity valuation of suitable open spaces used for OD (i.e., as the last resort option) owing to resource constraints preventing toilet adoption and the opportunity-driven OD hypothesis, which contends that it is the availability of suitable spaces per se that attracts people to defecate in the open. We geolocate 283 major OD sites in 175 villages of Purulia district (West Bengal, India), assess their environmental parameters, and propose the OD Suitability Index (ODSI). We linked the village-wise ODSI with social data from structured interviews in 1,240 households practicing OD to investigate the social valuation of the spatial environment concerning OD (specific type of sanitation ecosystem services). Results reveal that the perceived importance of the availability of suitable space for OD is negatively related to the ODSI measure, implying that people acknowledge the scarcity value of suitable space for OD due to the absence of other options rather than its abundance per se. Besides, we also showed that the perceived resource shortages have an objective basis and are negatively related to household income. These findings support the contention that the widespread occurrence of OD in the studied region is best explained by necessity-driven OD rather than the opportunity-driven OD argument. Thus, addressing the resource constraints should be prioritized over (prior to) sanctioning people practicing OD.

Molecular cytogenetics of four species of Calendula (Asteraceae)

For the first time, a comparative molecular cytogenetic study of four species of Calendula L. (Asteraceae): C. officinalis, C. stellata, C. tripterocarpa, and C. arvensis was carried out. In each species, chromosome numbers were determined: C. stellata (2n = 2х = 14), C. officinalis (2n = 4x = 32), C. tripterocarpa (2n = 2x = 30), and C. arvensis (2n = 4x = 44), and also specific chromosome localization of 45S and 5S rDNA clusters was revealed with the use of fluorescence in situ hybridization. An additional polymorphic minor 45S rDNA hybridization signal was found in the C. officinalis karyotype, which was located median in the short arm of one chromosome. The patterns of chromosome distribution of the major sites of 45S and 5S rDNA in karyotypes of the studied species confirmed the hybrid origin of C. arvensis (2n = 4x = 44) which could be a result of the introgressive hybridization of two other species: C. stellata (2n = 2x = 14) and C. tripterocarpa (2n = 2x = 30) during speciation.

Specific transport of temozolomide does not override DNA repair-mediated chemoresistance

Temozolomide (TMZ) a DNA alkylating agent, is the standard-of-care for brain tumors, such as glioblastoma multiforme (GBM). Although the physicochemical and pharmacokinetic properties of TMZ, such as chemical stability and the ability to cross the blood-brain barrier (BBB), have been questioned in the past, the acquired chemoresistance has been the main limiting factor of long-term clinical use of TMZ. In the present study, an L-type amino acid transporter 1 (LAT1)-utilizing prodrug of TMZ (TMZ-AA, 6) was prepared and studied for its cellular accumulation and cytotoxic properties in human squamous cell carcinoma, UT-SCC-28 and UT-SCC-42B cells, and TMZ-sensitive human glioma, U-87MG cells that expressed functional LAT1. TMZ-AA 6 accumulated more effectively than TMZ itself into those cancer cells that expressed LAT1 (UT-SCC-42B). However, this did not correlate with decreased viability of treated cells. Indeed, TMZ-AA 6, similarly to TMZ itself, required adjuvant inhibitor(s) of DNA-repair systems, O6-methylguanine-DNA methyl transferase (MGMT) and base excision repair (BER), as well as active DNA mismatch repair (MMR), for maximal growth inhibition. The present study shows that improving the delivery of this widely-used methylating agent is not the main barrier to improved chemotherapy, although utilizing a specific transporter overexpressed at the BBB or glioma cells can have targeting advantages. To obtain a more effective anticancer prodrug, the compound design focus should shift to altering the major DNA alkylation site or inhibiting DNA repair systems.

Cardiac nitric oxide scavenging: role of myoglobin and mitochondria.

Vascular production of nitric oxide (NO) regulates vascular tone. However, highly permeable NO entering the cardiomyocyte would profoundly impact metabolism and signalling without scavenging mechanisms. The purpose of this study was to establish mechanisms of cardiac NO scavenging. Quantitative optical studies of normoxic working hearts demonstrated that micromolar NO concentrations did not alter mitochondria redox state or respiration despite detecting NO oxidation of oxymyoglobin to metmyoglobin. These data are consistent with proposals that the myoglobin/myoglobin reductase (Mb/MbR) system is the major NO scavenging site. However, kinetic studies in intact hearts reveal a minor role (∼9%) for the Mb/MbR system in NO scavenging. In vitro, oxygenated mitochondria studies confirm that micromolar concentrations of NO bind cytochrome oxidase (COX) and inhibit respiration. Mitochondria had a very high capacity for NO scavenging, importantly, independent of NO binding to COX. NO is also known to quickly react with reactive oxygen species (ROS) in vitro. Stimulation of NO scavenging with antimycin and its inhibition by substrate depletion are consistent with NO interacting with ROS generated in Complex I or III under aerobic conditions. Extrapolating these in vitro data to the intact heart supports the hypothesis that mitochondria are a major site of cardiac NO scavenging. KEY POINTS: Cardiomyocyte scavenging of vascular nitric oxide (NO) is critical in maintaining normal cardiac function. Myoglobin redox cycling via myoglobin reductase has been proposed as a major NO scavenging site in the heart. Non-invasive optical spectroscopy was used to monitor the effect of NO on mitochondria and myoglobin redox state in intact beating heart and isolated mitochondria. These non-invasive studies reveal myoglobin/myoglobin reductase plays a minor role in cardiac NO scavenging. A high capacity for NO scavenging by heart mitochondria was demonstrated, independent of cytochrome oxidase binding but dependent on oxygen and high redox potentials consistent with generation of reactive oxygen species.

Mucosal response of inactivated and recombinant COVID-19 vaccines in Congolese individuals.

The efficacy of immunization against an airborne pathogen depends in part on its ability to induce antibodies at the major entry site of the virus, the mucosa. Recent studies have revealed that mucosal immunity is poorly activated after vaccination with messenger RNA vaccines, thus failing in blocking virus acquisition upon its site of initial exposure. Little information is available about the induction of mucosal immunity by inactivated and recombinant coronavirus disease 2019 (COVID-19) vaccines. This study aims to investigate this topic. Saliva and plasma samples from 440 healthy Congolese were collected including (1) fully vaccinated 2 month postvaccination with either an inactivated or a recombinant COVID-19 vaccine and (2) nonvaccinated control group. Total anti-severe acute respiratory syndrome coronavirus 2 receptor-binding domain IgG and IgA antibodies were assessed using in-house enzyme-linked immunosorbent assays for both specimens. Altogether, the positivity of IgG was significantly higher in plasma than in saliva samples both in vaccinated and nonvaccinated control groups. Inversely, IgA positivity was slightly higher in saliva than in plasma of vaccinated group. The overall IgG and IgA levels were respectively over 103 and 14 times lower in saliva than in plasma samples. We found a strong positive correlation between IgG in saliva and plasma also between IgA in both specimens (r = .70 for IgG and r = .52 for IgA). Interestingly, contrary to IgG, the level of salivary IgA was not different between seropositive control group and seropositive vaccinated group. No significant difference was observed between recombinant and inactivated COVID-19 vaccines in total IgG and IgA antibody concentration release 2 months postvaccination both in plasma and saliva. Inactivated and recombinant COVID-19 vaccines in use in the Republic of Congo poorly activated mucosal IgA-mediated antibody response 2 months postvaccination.

Вычислительный фантом для дозиметрии красного костного мозга пятилетнего ребенка от инкорпорированных бета-излучателей

The red bone marrow (RBM) exposure due to bone-seeking radionuclides can lead to grave medical consequences. In particular, the increased risk of leukemia in people exposed due to contamination of the Techa River in 1950s is associated with the RBM exposure due to 89,90Sr. Improvement of the internal RBM dosimetry methods includes the development of computational phantoms that represent 3D models of the skeletal sites. Modeling radiation transport within such phantoms enables estimation of conversion factors from the radionuclide activity in the bone to the RBM dose rate. This paper is an extension study focused on generating a set of computational phantoms representing skeletons of individuals of different ages. The aim was to develop a computational phantom representing a 5-yearold child for internal RBM dosimetry from incorporated beta emitters. The phantoms of the skeletal sites with active hematopoiesis were created using the original Stochastic Parametric Skeletal Dosimetry (SPSD) method. With this method, every such site represented a set of smaller phantoms of simple geometric shape. RBM distribution across the skeleton, bone size, characteristics of bone micro-architecture, as well as density and chemical composition of the simulated media (RBM, bone) were determined based on the published data. As a result, a computational phantom of the major skeletal sites with active hematopoiesis representing a 5-year-old child was generated that included 43 phantoms of bone fragments. Linear dimensions of phantoms were within 3–75 mm. Micro-architecture parameters varied greatly: BV/TV ratio —13–52%, Tb. Th. — 0.09–0.29 mm, Tb. Sp. —0.48–0.98 mm.

Expression of VP1 protein of foot-and-mouth disease virus serotype O originated from Indonesia in mammalian cells as potential immunogen

In 2022, Indonesia experienced foot-and-mouth disease (FMD) outbreak resulting in the death of hundreds of cattle; after more than 30 years, the country maintained FMD-free status. The FMD viral polypeptides-1 (VP1) protein contains major antigenic sites, making the protein have an important role in diagnostic applications or vaccine development. In this study, a synthetic codon-optimized vp1 gene from FMD virus serotype O was integrated into pcDNA3.1 vector and transformed to Escherichia coli TOP10F’ for plasmid propagation. The pcDNA3.1-1D-VP1 FMDV plasmid was then verified using agarose gel, showing in 659 bp size of the DNA fragment. The VP1 recombinant plasmid was transfected into HEK293T cells in DMEM medium supplemented with 10% FBS. Protein profiles were determined with SDS-PAGE showing target protein at 33KDa. Protein expression was confirmed by in-cell western assay using anti-VP1 type O polyclonal antibody and IRDye® 800CW goat anti-rabbit IgG as the secondary antibody. The result revealed a high-intensity fluorescence signal, indicating a strong interaction between expressed protein and anti-VP1 antibody. Thus, the results of this study demonstrate the potential for VP1 protein to be used as an immunogen in vaccine or diagnostic development against FMD infection. Nonetheless, some additional studies should be conducted.

Petrogenesis of the early Permian Hongliujing granite complex in the Chinese Eastern Tianshan orogen: Evidence for crustal growth in the Central Tianshan microcontinent

The generation and modification of silicic magma systems are essential processes in resolving the differentiation of continental crust. This understanding motivated the geochronological and geochemical study of the early Permian Hongliujing granite complex, consisting of quartz monzonite, granite, and leucogranite in the Central Tianshan microcontinent of the southern Central Asian Orogenic Belt. Laser ablation−inductively coupled plasma−mass spectrometry (LA-ICP-MS) zircon U-Pb dating of the Hongliujing complex rock units revealed almost identical ages (279 ± 2 Ma to 270 ± 2 Ma). The high-silica leucogranite and granite are characterized by positive Rb and negative Eu anomalies and Ba, Sr, P, and Ti depletions. The zircon trace elements are characterized by relatively low Ti and Th/U and high Yb/Gd. In contrast, the quartz monzonite and its mafic microgranular enclaves display minor negative Ba, Sr, P, Ti, and Eu anomalies, while the zircon trace elements are characterized by relatively high Ti and Th/U and low Yb/Gd. The complex has similar zircon Hf and whole-rock Nd isotopic compositions, with Hf and Nd model ages younger than 1.4 Ga, suggesting that their magmas were derived from an isotopically depleted mantle, with some contributions from crustal melts. The leucogranites further showed relatively large variations of εHf(t) and lower εNd(t) values, implying that their magma was affected by higher amounts of crustal contamination. We suggest that crystal-melt segregation was the major mechanism responsible for the evolution of the magmatic system, and that the early Permian magmatism represents a crust-forming episode triggered by slab rollback of the subducting South Tianshan oceanic plate beneath the eastern Central Tianshan microcontinent. Thus, our study reveals that microcontinents with Precambrian crustal basement were major sites of juvenile continental growth during the accretionary evolution of the Central Asian Orogenic Belt.

Evidence of Considerable Shifts in Catch Composition in the Artisanal Spiny Lobster Fishery in Kenya.

The artisanal lobster fishery in Kenya is small in world terms but important locally both in terms of supporting local fishing communities and generating revenue for the government. Despite its socioeconomic importance, detailed knowledge of many aspects of the fishery is lacking. The study reported herein aimed to investigate and provide information on the population structure and catch composition of spiny lobsters caught by artisanal fishers off six major landing sites along the coastline. A total of 2711 lobsters representing five palinurid species were collected during the study period (November 2000-March 2001). Among the five species, Panulirus longipes dominated the catches in Msambweni (75%) and Shimoni (58%), P. homarus in Mambrui (70%) and Kipini (72%), P. ornatus in Lamu (49%), and P. penicillatus in Kilifi (39%). P. versicolor was the rarest species observed in the catches across the six sites. The overall catch consisted of 33% P. ornatus, 32% P. homarus, 28% P. longipes, 6% P. penicillatus and 2% P. versicolor. Sitewise, Lamu contributed 31% of the overall catch, Kipini 23%, Shimoni 20%, Mambrui 13%, Msambweni 7%, and Kilifi 6%. A comparison of the results of this study and lobster abundance data from 1970s surveys revealed considerable shifts in the catch composition of artisanal landings over time. Future work on this fishery should concentrate on the lobster populations in the decades-old marine protected areas to obtain unfished reference data to assess the fishery and establish the underlying cause(s) of the observed shifts in catch composition.

Checkpoint Inhibitor PD-1H/VISTA Affects Myeloma Bone Disease By Osteoclast Cytoskeleton Remodeling

Introduction We have recently described the critical role of checkpoint inhibitor PD-1H/VISTA in osteoclast activation and multiple myeloma bone disease. Highly expressed in osteoclast precursors and mature osteoclasts, PD-1H functions as the receptor for osteoclastogenic factor MMP-13 and mediates MMP-13 induced osteoclast activation and myeloma bone disease ( Nat Commun14, 4271 (2023)). Despite the extensive functional study of PD-1H in immune disease models, the PD-1H interacting cellular complex is yet to be defined. Here we report how PD-1H interacts with cytoskeleton proteins contributing to osteoclast remodeling in multiple myeloma bone disease. Methods and Results To identify interacting proteins of PD-1H, PD-1H-His 6 recombinant protein was overexpressed in mouse mononuclear BMCs, and PD-1H-His 6 binding protein complex was pulled down by Ni-NTA agarose beads followed by mass spectrum analysis (Figure A). LIM and SH3 domain protein 1 (LASP1) was one of the most abundant cytoskeleton proteins identified in the PD-1H complex. We therefore first confirmed its binding to PD-1H by two-way co-IP assays (Figure B and C). Co-localization of LASP1 and PD-1H were further confirmed by immunofluorescence assay when co-expressed in HEK293 cells (Figure D). LASP1 is an F-actin-binding protein that stabilizes F-actin bundles and is involved in podosome regulation in macrophages (PLoS ONE 7(4): e35340). It has four domains: LIM, two nebulin-like repeats (N1 and N2) and SH3 domains. While nebulin-like repeats mediate binding to F-actin, SH3 domain is the major protein-protein binding site (PLoS ONE 7(4): e35340). In co-IP assay using PD-1H and domain deleted LASP1, deletion of LIM (△LIM) or either one or both nebulin-like repeats (△N1, △N2 and △N1/2) did not affect the binding to PD-1H. Instead, deletion of SH3 (△SH3) in LASP1 lead to loss of PD-1H binding, demonstrating that PD-1H binds to LASP1 at the SH3 domain (Figure E). Next, the association of LASP1 and PD-1H in the osteoclast cytoskeleton was demonstrated by confocal immunofluorescence microscopy. LASP1 co-localized with F-actin (Figure F) and PD-1H (Figure G) at the sealing ring in mature osteoclasts. Interestingly, although MMP-13 stimulation increased the size of osteoclasts and the sealing ring, MMP-13 did not change the co-localization pattern of LASP1 with PD-1H or F-actin. Conclusions Taken together, this study reveals the novel mechanism of PD-1H regulation of osteoclasts cytoskeleton reorganization, which is critical for osteoclast bone resorption activity. PD-1H directly associates with LASP1 at its C-terminal SH3 domain. Although LASP1 was reported to regulates podosomes function in macrophages, its role in osteoclast cytoskeletons remain largely obscure. The future study will be focused on delineating the role of LASP1 in osteoclast function, and its role in MMP-13/PD-1H signaling regulated myeloma bone disease.

Intracellular Iron Controls HSC Metabolism By Affecting Mitochondrial Fitness

Iron is an important source of reactive oxygen species (ROS) within the cell and recent evidence highlighted its role in regulating HSC self-renewal and differentiation. However, whether and how iron influences HSC metabolism is still undetermined. Cellular metabolism is a key regulator of HSC maintenance and HSCs adapt their metabolic state to their function. Quiescent HSCs have low energy requirements and depend on anaerobic glycolysis, whereas active HSCs require high energy for proliferation and differentiation thus enhancing oxidative phosphorylation (OXPHOS), glycolysis and fatty acid oxidation (FAO). To explore which metabolic pathways are triggered by intracellular iron levels, we took advantage of the thalassemic Hbb th3/+mice ( th3), characterized by chronic iron overload (IO). We previously demonstrated an impaired function of th3 HSCs due to persistence into altered BM niche ( Aprile et al., 2020). Th3 HSCs showed higher levels of intracellular free reactive iron (1.7±0.2 calcein MFI fold to wt, p<0.0001) and interestingly iron specifically accumulated in the mitochondria. As a result, mitochondria are impaired, with low mass and activity, as assessed by decreased mitochondrial membrane potential ( th3 1068±167 vs. wt 3215±686 MFI TMRE, p<0.05). These data, together with a reduced expression of mitochondrial biogenesis and mitophagy genes, indicate an accumulation of damaged mitochondria. To demonstrate a direct effect of iron on mitochondrial fitness in HSCs, we treated wt mice with iron dextran. Consistently, IO wt HSCs showed reduced mitochondrial activity following intracellular iron accumulation. Since mitochondria are the major site of cellular ATP production mostly through OXPHOS, we measured the metabolic state of HSCs in ß-thalassemia. ATP levels in sorted th3 HSCs was halved than the one in wt mice ( th3 3.7±0.3 vs. wt 9.8±2.6 ATP nM, p<0.01). Inhibition of OXPHOS by oligomycin lowered the ATP content in wt cells with a minor effect in th3 HSCs, thus indicating a reduced OXPHOS-dependent metabolism, despite their active cycling profile. Since mitochondria are damaged and OXPHOS and glycolysis are the two main metabolic pathways involved in cellular ATP generation, we hypothesized increased glycolytic dependency to compensate for reduced OXPHOS in th3 HSCs. Indeed, in these cells we observed a positive enrichment of glycolytic genes, such as Tpi1, Gapdh and Pklr, and a 1.4-fold higher glucose uptake ( th3 2401±85 vs. wt 1639±163 MFI 2NBDG, p<0.05). In vivo administration of the iron chelator DFO normalized intracellular iron content and rescued mitochondrial dysfunction in th3 mice, indicating the reversibility of mitochondrial defects. Moreover, HSCs from DFO-treated mice showed reduced glucose uptake, thus demonstrating that IO causes mitochondrial defects and metabolic adaptations of HSCs. Excess iron is expected to give rise to ROS and, consistently, th3 HSCs displayed high mitochondrial ROS (mtROS) levels ( th3 5205±459 vs. wt 2507±756 MFI MitoSOX, p<0.01). I n vivo reduction of mtROS by MitoQ administration restored mitochondrial activity and decreased glucose uptake. Remarkably, MitoQ rescued the quiescence and self-renewal ability of th3 HSCs, as shown by superior chimerism in secondary transplantation of HSCs from MitoQ-treated th3 donors in lethally irradiated recipients ( th3 HSC+MitoQ 81.7±1.6 vs. untreated th3 HSC 34.9±14.6 vs. % of chimerism, p<0.05), thus indicating that IO-derived ROS impair HSC function by affecting mitochondrial fitness and bioenergetics. Preliminary results confirmed intracellular IO and mitochondrial defects also in HSCs from Townes mice, a model of sickle cell anemia characterized by IO. Collectively, our study revealed that increased intracellular iron and ROS levels alter HSC metabolic programs by inducing mitochondrial dysfunction. Since mitochondria are defective, IO HSCs are unable to produce appropriate levels of ATP through OXPHOS and rely on glycolysis, despite their active cycling activity. Finally, the rescue of mitochondrial activity can restore the functional defects of th3 HSCs.

Iron Chelation Efficiency in Human Hepatocytes Is Enhanced By Exogenous Hepcidin

Iron chelation is clinically inefficient, with only a small percentage of the applied chelator being excreted in the iron-bound form. Chelation is also slow because only a small proportion of storage iron is in the chelatable labile iron pool (LIP) at any given moment. In principle, by increasing the magnitude of the LIP within hepatocyte, the major site of body storage iron in conditions of iron overload, the magnitude and hence the efficiency of chelation may be increased. Recent work has shown that SLN124, a RNAi-therapeutic inhibitor of Tmprss6 gene expression, increases iron chelation by deferiprone in a murine model of thalassemia (b-thalassemic Hbb th3/+ animals) (Vadolas et al Brit J Haematol 2021) and murine hereditary haemochromatosis (Altamura et al. HemaSphere 2019). We wished to determine whether the mechanism of increased chelation was consistent with the blocking of iron exit from hepatocyte through ferroportin channels by increased levels of hepcidin. Here we have examined whether application of exogenous hepcidin to a human hepatocyte cell line HepG2 can increase the chelation efficiency of deferiprone and whether this effect is also seen with other iron chelators such as deferasirox and deferoxamine. A model was developed from previous work (Vlachodimitropoulou et al. Blood 2017) where the net effect of iron uptake (from fetal bovine serum medium) versus intracellular iron removal by chelation, could be interrogated. The dose and time-dependent effect of exogenous hepcidin and its interaction with iron chelators on intracellular iron content was examined. Briefly, to load cells with iron, confluent HepG2 cells were incubated for 2h with 10% FBS followed by media change containing 10-30 uM iron binding equivalents (IBE) of therapeutic chelators, hepcidin, or control media. This loading method doubled the cellular iron; and subsequently increased cellular ferritin by at least 4-fold. Both effects could be completely inhibited with iron chelation. Following a pulse with hepcidin (100nM-10µM), intracellular iron content of HepG2 cells increased in a dose dependent manner, by 30-100% over the next 4h, followed by a decrease in cellular iron at 8 hours (Fig. 1A). This transient effect on iron retention could be prolonged with serial addition of hepcidin to the media (at 2 and 4h, data not shown). Co-treatment with the exclusively extracellular chelator CP40 demonstrated that while a substantial fraction of the hepcidin effect on iron retention was dependent on iron uptake from media (Fig 1B), the remaining increase in iron retention was independent of this effect and consistent with inhibition of iron egress from . Compared with control cells (without chelator but with or without hepcidin), a 50-200% enhancement of hepatocyte iron removal by 10uM IBE deferiprone was observed at 2 and 4 hours (Fig. 1C) with the addition of 300nM exogenous hepcidin. Correspondingly, cellular iron removal with deferoxamine (10-30 µM IBE) was enhanced by 250% at 4 h by exogenous hepcidin (Fig. 1D) whereas iron removal by deferasirox (30µM IBE) was enhanced by at least 50% at 2 and 4 h when hepcidin (300nM) was present (Fig. 1E), as compared to controls. Enhanced chelation with DFO was not observed until after 2h incubation, consistent with the known slower access of DFO to intracellular pools compared with deferiprone or deferasirox. Beyond 4h, the effect of hepcidin on enhanced chelation was not seen, suggesting that hepcidin is consumed or degraded in this culture system. We conclude that enhanced removal of intracellular iron by chelation is demonstrable with all three clinically available chelators following the application of exogenous hepcidin. This iron would be destined for increased excretion of chelate complexes in vivo and hence enhanced decrements in body iron. Two mechanisms for enhanced chelation are possible in this model; the first is by increased iron loading of hepatocytes incubated with hepcidin and secondly through the blocking by hepcidin of labile intracellular iron egress through ferroportin channels, thereby increasing the magnitude of chelatable intracellular iron pools. Future work in primary human hepatocytes will examine whether manipulation of intracellular hepcidin levels by SLN124 has similar chelation-enhancing effects.

Hspa5 Deficiency Blocks Intestinal Enterocyte Differentiation in Graft-Versus-Host Disease

Background: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been established as an effective therapy for hematological malignancies. Graft-versus-host disease (GVHD) is a life-threatening complication, and the intestine is one of the major sites affected. Currently, immunosuppressive agents remain the preferred choice for GVHD treatment. However, treatment resistance still occurs in a large population, indicating that some tissue damage may be irreversible. Methods: The murine transplant and GVHD model were established as previously described. To characterize the intestinal damage mediated by radiation and GVHD, murine small intestine tissues were collected at day 14 after transplantation. Primary small intestinal crypts were isolated from each group and cultured for five consecutive days to evaluate their ability to proliferate and differentiate. Single-Cell analysis was performed to characterize the impaired intestinal repair after injury. Results: At day 14, the villus-to-crypt ratio was partially recovered in the BM group, but remained imbalanced in the GVHD group. We observed a significant decrease in the expression of Reg1, a gene expressed by enterocytes (EC), in the GVHD groups compared to the BM group. we further utilized a 3D organoid model to investigate intestinal growth and differentiation. The organoid size from the GVHD group was comparable to the BM and control groups. However, the number of buds per organoid was significantly reduced in the GVHD group compared to the BM and control groups. Immunofluorescence staining confirmed that cells in GVHD-derived organoids exhibited high proliferation (stained by Mki67) but impaired differentiation of enterocyte (stained by Aldob). To gain insights into the cellular mechanisms influencing enterocyte differentiation, we dissociated murine small intestinal tissues from each group into single cells and sequenced them using the 10X Genomics platform. Through pseudochronological analysis, each cell was ordered along the predicted lineages and assigned a pseudotime. We found that enterocytes in GVHD exhibited a long and discontinuous trajectory, suggesting explicit blockage of EC differentiation. Functional enrichment analysis showed that pathways related to cell growth and differentiation, such as MAPK, ERBB, and inositol phosphate metabolism, were highly enriched in M2 of the control and BM groups. In contrast, MAPK was dramatically downregulated at the early stage of ISC differentiation in the GVHD group. Next, we analyzed the differentially expressed genes in EP between the GVHD and BM groups. Endoplasmic reticulum (ER) chaperone-related genes from the heat shock protein (Hsp) 70 family (Hspa1a/1b/5), Hsp90 family (Hsp90aa1/ab1), and Hsp40 family (Dnajb1) were significantly decreased in GVHD compared to BM. Notably, Hspa5 (also known as GRP78 or Bip), which serves as a sensor of glucose levels and misfolded proteins, exhibited reduced protein levels, along with decreased IRE1ɑ phosphorylation (p-IRE1ɑ) and Xbp1s, as well as decreased PERK phosphorylation (p-PERK) and ATF6 expression. To validate our conjecture, we utilized small intestinal organoids cultured for 3 days for further experimentation. RT-qPCR demonstrated that energy restriction downregulated the mRNA expression of Hspa5 and other chaperones. Furthermore, we treated intestinal organoids with HA15, a BIP inhibitor, which resulted in significantly enlarged area but fewer buds. Consistent with our previous findings, Hspa5 was downregulated in the HA15-treated group, along with decreased expression of Hsp family and enterocyte markers. Since Hsp family plays key roles in restoring cellular homeostasis, we wondered if upregulating Hspa5 could protect intestinal cells from outside stimuli such as pro-inflammatory cytokines. When intestinal organoids were cocultured with IFN-g, we found that Bix, the selective Bip inducer, protected organoids from apoptosis by increasing Hsp70 expression. Conclusion: Given that immunosuppression partly resolves GVHD, strategies aimed at restoring intestinal homeostasis should be given sufficient attention. Our study reveals that intestinal enterocyte differentiation is blocked in GVHD, possibly due to starvation and energy restriction. Deficiency in Hspa5 results in impaired intestinal repair, and increasing Hsp levels may enhance cell resistance to cytokine storms.

Optimizing shared recreational vehicle service areas: A multi-strategy approach for economic performance and user satisfaction

In order to reduce overtourism and traffic congestion, local authorities may have to divert recreational vehicle traffic to off-site service areas. The problem that will arise is how best to accommodate different types of users with opposing preferences in the same area, some wanting to be as close as possible to the major site to be visited, others seeking peace and quiet. We have represented their specific attitudes using a two-stage decision-making process via a conjunctive model followed by a compensatory model. We then propose to model three strategies, seeking either to optimise customer attractiveness, or profit, or space occupation, in order to define a location, capacity and price for this shared area. Using a realistic data set, the results show that economic performance follows a concave curve as a function of the population mix. Moreover, only the strategy of maximising attractiveness suggests always mixing users in the same area.

The secreted peptide BATSP1 promotes thermogenesis in adipocytes

Although brown adipose tissue (BAT) has historically been viewed as a major site for energy dissipation through thermogenesis, its endocrine function has been increasingly recognized. However, the circulating factors in BAT that play a key role in controlling systemic energy homeostasis remain largely unexplored. Here, we performed a peptidomic analysis to profile the extracellular peptides released from human brown adipocytes upon exposure to thermogenic stimuli. Specifically, we identified a secreted peptide that modulates adipocyte thermogenesis in a cell-autonomous manner, and we named it BATSP1. BATSP1 promoted BAT thermogenesis and induced browning of white adipose tissue in vivo, leading to increased energy expenditure under cold stress. BATSP1 treatment in mice prevented high-fat diet-induced obesity and improved glucose tolerance and insulin resistance. Mechanistically, BATSP1 facilitated the nucleocytoplasmic shuttling of forkhead transcription factor 1 (FOXO1) and released its transcriptional inhibition of uncoupling protein 1 (UCP1). Overall, we provide a comprehensive analysis of the human brown adipocyte extracellular peptidome following acute forskolin (FSK) stimulation and identify BATSP1 as a novel regulator of thermogenesis that may offer a potential approach for obesity treatment.

Music and Storytelling at Rock Art Sites? The Archaeoacoustics of the Urkosh Area (Russian Altai)

Abstract In this article, the potential of archaeoacoustics for understanding past communities is discussed by looking at a range of acoustic parameters. Our case study is the Urkosh rock art area in the Ongudai district, Republic of Altai (Russia). The rock art of this area dates possibly from the Upper Palaeolithic and definitely from the Early Bronze Age (second half of the third millennium BCE). There are important periods in the Early Iron Age (first half of the first millennium BCE) and the medieval era, after which there are later additions up to the present day. Major and minor sites were tested, as well as some with no art. The results obtained from the tests conducted using the impulse response method indicate high values for sound clarity not only in the rock art sites but also in at least one nearby panel without rock art. Although these results cannot explain why rock art was produced precisely in specific locations, they objectively describe the acoustic conditions under which particular intangible cultural practices were probably organised in them. In particular we focus on storytelling and music, cultural practices for which there is a wealth of information in the ethnographic sources written about the area.

Cancer mortality patterns in selected Northern and Southern African countries.

Non-communicable diseases have been rapidly increasing in African countries. We provided updated cancer death patterns in selected African countries over the last two decades. We extracted official death certifications and population data from the WHO and the United Nations Population Division databases. We computed country- and sex-specific age-standardized mortality rates per 100 000 person-years for all cancers combined and ten major cancer sites for the periods 2005-2007 and 2015-2017. Lung cancer ranked first for male cancer mortality in all selected countries in the last available period (with the highest rates in Réunion 24/100 000), except for South Africa where prostate cancer was the leading cause of death (23/100 000). Prostate cancer ranked second in Morocco and Tunisia and third in Mauritius and Réunion. Among Egyptian men, leukemia ranked second (with a stable rate of 4.2/100 000) and bladder cancer third (3.5/100 000). Among women, the leading cancer-related cause of death was breast cancer in all selected countries (with the highest rates in Mauritius 19.6/100 000 in 2015-2017), except for South Africa where uterus cancer ranked first (17/100 000). In the second rank there were colorectal cancer in Tunisia (2/100 000), Réunion (9/100 000) and Mauritius (8/100 000), and leukemia in Egypt (3.2/100 000). Colorectal and pancreas cancer mortality rates increased, while stomach cancer mortality rates declined. Certified cancer mortality rates are low on a global scale. However, mortality rates from selected screening detectable cancers, as well as from infection-related cancers, are comparatively high, calling for improvements in prevention strategies.