Hypoxic Conditions Research Articles

Expression of pyruvate kinase, malate and octopine dehydrogenase genes in the gills of the Mediterranean mussel Mytilus galloprovincialis (Lamarck, 1819) under conditions of hypoxia and reoxygenation

The effect of hypoxia on the expression level of pyruvate kinase (PKM), octopine dehydrogenase (OcDH) and malate dehydrogenase (MDH) genes in the gill tissue of the Mediterranean mussel Mytilus galloprovincialis (L., 1819) was studied experimentally. The control group of mollusks was kept at 9–10°C and the oxygen level in the water was 8.5 mgO2 l–1. Experimental – at 9–10°C and 2.2 mgO2 l–1. The exposure was 24 and 72 hours. Some of the individuals were subsequently subjected to the reoxygenation procedure. Under conditions of hypoxia, the expression level of OcDH and MDH genes increased by 3 and 2 times, respectively (p 0.05). At the same time, OcDH expression showed sensitivity to the oxygen content in the medium. Under the conditions of reoxygenation, the process was completely suppressed. The expression of the PKM gene, on the contrary, did not depend on hypoxic effects and remained at the level of control values.

Effects of endurance exercise under hypoxic conditions on the gastric emptying rate and intestinal cell damage.

The present study examined the effects of gastric emptying rate and intestinal cell damage following a single session of endurance exercise under "hypoxic" or "normoxic" conditions at the same relative intensity. Eleven healthy males performed two trials on different days, consisting of a 60min run on a treadmill at 70% maximal running velocity (vMax) while inspiring hypoxic (FiO2: 14.5%; HYP) or normoxic air (FiO2: 20.9%; NOR). The average running velocity was 11.4 ± 0.7km/h in NOR and 10.8 ± 0.5km/h in HYP, respectively. Venous blood samples were collected to evaluate plasma intestinal fatty acid binding protein (I-FABP) as an indicator of exercise-induced intestinal cell damage. The gastric emptying rate was determined by the 13C-sodium acetate breath test. Running velocities at 70% vMax and arterial oxygen saturation were significantly lower under HYP than NOR (p < 0.001). Peak heart rate and rating of perceived exertion during exercise did not differ significantly between the trials. Maximum 13C excretion time (an indication of the gastric emptying rate) was significantly delayed in the HYP (NOR: 38.5 ± 5.0min, HYP: 45.5 ± 9.6min; p = 0.010). Furthermore, the score of nausea increased slightly, but increased significantly after exercise only in the HYP (p = 0.04). However, exercise-induced changes in plasma I-FABP, adrenaline, and noradrenaline concentrations did not differ significantly between the two trials. These results suggest that endurance exercise under hypoxic conditions impairs digestive function in the stomach compared to exercise under normoxic conditions performed at the same relative intensity.

Chronic Hypoxia in an EXTrauterine Environment for Neonatal Development Impairs Lung Development.

Severe fetal hypoxia poses a significant risk to lung development resulting in severe postnatal complications. Existing chronic hypoxia animal models lack the ability to achieve pathologically reduced fetal oxygen without compromising animal development, placental blood flow, or maternal health. Using an established model of isolated chronic hypoxia involving the Extrauterine Environment for Neonatal Development (EXTEND), we are able to investigate the direct impact of fetal hypoxia on lung development. Oxygen delivery to preterm fetal lambs (105-110 days GA) delivered by cesarean section was reduced, and animals were supported on EXTEND through the canalicular or saccular stage of lung development. Fetal lambs in hypoxic conditions showed significant growth restriction compared to their normoxic counterparts. We also observed modest aberrant vascular remodeling in the saccular group after hypoxic conditions with decreased macrovessel numbers, microvascular endothelial cell numbers, and increased peripheral vessel muscularization. In addition, fetal hypoxia resulted in enlarged distal airspaces and decreased septal wall volume. Moreover, there was a reduction in mature SFTPB and processed SFTPC protein expression concomitant with a decrease in AT2 cell number. These findings demonstrate that maternally-independent fetal hypoxia predominantly impacts distal airway development, AT2 cell number, and surfactant production with mild effects on the vasculature.

The Effect of Pre-Exercise Caffeine and Glucose Ingestion on Endurance Capacity in Hypoxia: A Double-Blind Crossover Trial.

The impact of caffeine and glucose supplementation in a hypoxic environment on endurance exercise performance remains inconclusive. The current study examined the effect of pre-exercise carbohydrate and caffeine supplementation on endurance exercise performance in an acute hypoxic environment. Eight healthy active young males participated in this double-blind, within-subjects crossover study. Participants ingested the test drink 60 min before exercising at 50% Wmax for 90 min on a cycle ergometer (fatiguing preload); there followed an endurance performance test at 85% Wmax until exhaustion in a hypoxic chamber (~15%O2). Participants completed four experimental trials in a randomized order: caffeine (6 mg·kg-1; Caff), glucose (1 g·kg-1; CHO), caffeine (6 mg·kg-1) + glucose (1 g·kg-1; Caff-CHO), and taste- and color-matched placebo with no caffeine or CHO (PLA). Blood samples were collected during fasting, pre-exercise, every 30 min throughout the exercise, and immediately after exhaustion. The caffeine and glucose trials significantly enhanced endurance capacity in hypoxic conditions by Caff, 44% (68.8-31.5%, 95% confidence interval), CHO, 31% (44.7-15.6%), and Caff-CHO, 46% (79.1-13.2%). Plasma-free fatty-acid and glycerol concentrations were higher in Caff and PLA than in CHO and Caff-CHO (p < 0.05). The estimated rate of fat oxidation was higher in Caff and PLA than in CHO and Caff-CHO (p < 0.05). There were no significant differences in ratings of perceived exertion between trials. In conclusion, the ingestion of caffeine, glucose, or caffeine + glucose one hour before exercising in hypoxic conditions significantly improved 85% Wmax endurance performance after prolonged exercise.

Chronic hypoxia for the adaptation of extracellular vesicle phenotype

Variations in oxygen level affect the phenotype of cells and extracellular vesicles (EVs). Depending on the metabolic oxygen demand of cells, hypoxic cell culture can produce conditions more like those found in vivo, and with appropriate oxygen levels, mimic hypoxic tumours. However, most previous experiments studying both EVs and the effects of hypoxia on cells use periods of 72 h or less of hypoxia. We hypothesised that this was insufficient time for adaptation to hypoxic conditions both for EVs and cells which may skew the results of such studies. In this study, the effects of acute (72 h) and chronic hypoxia (> 2 weeks) on the phenotype of HepG2 and PC3 cells and their EVs were examined. Cells could be cultured normally under chronic hypoxic conditions and cryopreserved and recovered. The effects of hypoxia on EV phenotype are slow to establish and dependent on cell line. In PC3 cells, the greatest change in phenotype and increase in EV production occurred only with chronic hypoxic culture. In HepG2 cells, the number of EVs produced was insensitive to hypoxic culture and the greatest changes in protein expression were observed after acute hypoxic culture. Nonetheless, biphasic changes in EV phenotype were detected in both cell types in response to either acute or chronic hypoxia. These results indicate that for cells which do not induce consumptive oxygen depletion, prolonged hypoxic culture is required for complete adaptation.

Endothelin-1 potentiated constriction in preeclampsia placental veins: Role of ETAR/ETBR/CaV1.2/CALD1

BackgroundPlacenta plays a vital role in preeclampsia. The present study investigated the role of endothelin-1 (ET-1) and its receptors in preeclampsia placenta. MethodPlacenta samples were collected from normal and preeclampsia pregnancies, with one single fetus. Placental chorionic plate vessel tone was measured with DMT using vasoactive agents with or without antagonists. Role of L-type voltage-dependent calcium channels (CaV1.2) in single smooth muscle cell was detected using whole-cell patch clamp. PCR, Western blot, and ELISA was used to detect molecule expressions. Placental vessel explants and human umbilical vein smooth muscle cell (HUVSMC) were exposed to ET-1 treatment with or without antagonists. Human umbilical vein endothelial cell (HUVEC) and pregnant sheep was exposed to hypoxic condition, simulating preeclampsia. ResultsET-1 and IRL1620 mediated stronger contractions in preeclampsia placental veins, despite unchanged ETAR and decreased ETBR expression. Comparing with control, there was higher ET-1 in umbilical plasma, maternal plasma, and placental vessels from preeclampsia. In utero hypoxia increased plasma ET-1 in fetal lambs and ewes. Hypoxia promoted ET-1 production in HUVEC. Role and expression of CaV1.2 was decreased in preeclampsia placental vessels, while high-molecular-weight caldesmon (CALD1), the marker of contractile phenotype of smooth muscle cells, was significantly increased. ET-1 treatment increased CALD1 in placental explants and in HUVSMC via ETAR/ETBR. ConclusionThe present study firstly demonstrated ET-1 induced greater contraction in preeclampsia placental chorionic plate veins via ETAR/ETBR, instead of via weaker CaV1.2. In utero hypoxia promoted plasma ET-1 in fetal lambs and ewe, similar to that in preeclampsia. ET-1, binding with ETAR/ETBR increased CALD1, which was associated with stronger contraction in preeclampsia. The data provided important information in preeclampsia onset.

Chlorella-enriched hydrogels protect against myocardial damage and reactive oxygen species production in an in vitro ischemia/reperfusion model using cardiac spheroids

Microalgae have emerged as promising photosynthetic microorganisms for biofabricating advanced tissue constructs, with improved oxygenation and reduced reactive oxygen species (ROS) production. However, their use in the engineering of human tissues has been limited due to their intrinsic growth requirements, which are not compatible with human cells. In this study, we first formulated alginate-gelatin (AlgGel) hydrogels with increasing densities ofChlorella vulgaris. Then, we characterised their mechanical properties and pore size. Finally, we evaluated their effects on cardiac spheroid (CS) pathophysiological response under control and ischemia/reperfusion (I/R) conditions. Our results showed that the addition ofChlorelladid not affect AlgGel mechanical properties, while the mean pore size significantly decreased by 35% in the presence of the 107cells ml-1microalgae density. Under normoxic conditions, the addition of 107Chlorellacells ml-1significantly reduced CS viability starting from 14 d in. No changes in pore size nor CS viability were measured for hydrogels containing 105and 106Chlorellacells ml-1. In our I/R model, allChlorella-enriched hydrogels reduced cardiac cell sensitivity to hypoxic conditions with a corresponding reduction in ROS production, as well as protected against I/R-induced reduction in cell viability. Altogether, our results support a promising use ofChlorella-enriched Alg-Gel hydrogels for cardiovascular tissue engineering.

Introduction of asingle-nucleotide variant, rs16851030, into the ADORA1 gene increased cellular susceptibility to hypoxia.

Aim: Rs16851030, a single-nucleotide variant located in the 3'-untranslated region of the ADORA1 gene, has been proposed as a potential marker of caffeine sensitivity in apnea of prematurity. Besides, it is associated with aspirin-induced asthma and the development of acute chest syndrome. However, its functional significance is still unconfirmed. This study aimed to elucidate the functional impact of rs16851030 by using CRISPR/Cas9 approach to induce the DNA variant and attendant physiological changes.Methods: Rs16851030 was introduced into HEK293 cells via homology-directed repair (HDR). Edited cells were fluorescence-enriched, sorted, isolated, and expanded into single-cell-derived clones. The edit was confirmed by Sanger sequencing. RNA sequencing was used to analyze affected pathways.Results: Rs16851030-mutant cells showed increased susceptibility to hypoxia, a condition related to apnea of prematurity. After 24h of hypoxia, the viability of mutant clones 1 and 2 was low compared with wild-type cells (75.45% and 74.47% vs. 96.34%). RNA sequencing revealed transcriptomic changes linked to this increased vulnerability.Conclusion: Rs16851030 impairs cellular resistance to hypoxia, suggesting its role in conditions like apnea of prematurity. Further research should investigate the molecular mechanisms and transcriptomic alterations caused by rs16851030 under hypoxic conditions.

Candida glabrata maintains two HAP1 ohnologs, HAP1A and HAP1B, for distinct roles in ergosterol gene regulation to mediate sterol homeostasis under azole and hypoxic conditions.

Invasive and drug-resistant fungal infections pose a significant public health concern. Candida glabrata, a human fungal pathogen, is often difficult to treat due to its intrinsic resistance to azole antifungal drugs and its capacity to develop clinical drug resistance. Therefore, understanding the pathways that facilitate fungal growth and environmental adaptation may lead to novel drug targets and/or more efficacious antifungal therapies. While the mechanisms of azole resistance in Candida species have been extensively studied, the roles of zinc cluster transcription factors, such as Hap1A and Hap1B, in C. glabrata have remained largely unexplored until now. Our research shows that these factors play distinct yet crucial roles in regulating ergosterol homeostasis under azole drug treatment and oxygen-limiting growth conditions. These findings offer new insights into how this pathogen adapts to different environmental conditions and enhances our understanding of factors that alter drug susceptibility and/or resistance.

Oxygenating respiratoid biosystem for therapeutic cell transplantation

In this study, we address the persistent challenge of providing adequate oxygen to transplanted cells by introducing a respiratoid biosystem. Central to our strategy is the chloroplast-transit-peptide (CTP), crucial for optimal oxygenation. Through conjugation of CTP with alginate, we achieve stabilization of chloroplast structure. Strategically anchored to the outer chloroplast membrane, CTP not only ensures structural integrity but also upregulates key photosynthesis-associated genes. This biosystem demonstrates exceptional efficacy in spontaneously generating oxygen, particularly under hypoxic conditions (~1% pO2). In an application, pancreatic islets encapsulated within the respiratoid biosystem and intraperitoneally implanted in diabetic mice maintain normal glucose levels effectively. Insulin secretion persists for 100 days post-xenotransplantation without the need for immunosuppressant administration, highlighting the reliance on the respiratoid biosystem’s oxygen supply and structural stability. Our study demonstrates the respiratoid biosystem as a platform in tissue engineering, offering a nature-inspired solution to the critical challenge of spontaneous oxygen supply.

Novel anti-tumor effect of natural products from Aloe vera resin and their in-vitro/in-silico targeting mechanism of carbonic anhydrase-II and IX.

Human carbonic anhydrase (hCA) plays a vital role in the development and progression of tumors in hypoxic conditions. Herein we report the hCA-II and hCA-IX activities of natural products isolated from Aloe vera (L.) Burm.f., to know their potential in tumors. These isolated compounds (1-10) displayed varying degrees of inhibition against hCA-II and hCA-IX. All the compounds showed potent activity against hCA-IX with IC50 values in the range of 2.9 - 29.1 µM. While for hCA-II, compounds 1, 2, 5-10 exhibited IC50 in the range of 4.7 - 23.4 µM. The most effective hCA IX and II inhibitors, 2 and 5, were chosen for in vitro mechanism studies, revealing that they are competitive inhibitors. Furthermore, when tested for their cytotoxic effect on BJ (normal) cell line, all the compounds showed no cytotoxic behavior, while on Prostate cancer cells (PC-3), compounds 1, 3, 5, 7, and 9 exhibited significant antiproliferative activity. Molecular docking was also conducted within the hCA IX and hCA-II active sites to observe their binding capability. Compounds 1, 5, 7, and 9 were active against both isozymes of hCA and in the PC-3 cell line, therefore these are the best choices for further in vivo studies..

Modified perylene diimide for femto molar level detection of glucose: smartphone-assisted colorimetric glucose detection kits.

In this report, a functionalized hydroxyphenyl benzothiazole (HBT) derivative has been synthesized and anchored onto the perylene diimide (PDI) core at the -bay position (PHI). PHI has been explored for the generation of radical anions (PH1˙-) and dianions (PH12-) in 20% HEPES buffer-DMSO solution using H2S as a sacrificial electron donor. The PH1˙- has a half-life (t1/2) of 1.5 h and 3 h in oxygenated and hypoxic conditions, respectively. The formation of radical anions has been confirmed by optical (absorbance and fluorescence) methods, cyclic voltammetry (CV), differential pulse voltammetry (DPV), and femtosecond transient absorbance spectroscopy along with current-voltage (I-V) and NOBF4 studies. The PH1˙- showed peroxidase-like activity for the reduction of H2O2 as low as 170 fmol L-1 (fM) giving a colour change from sea green to pink. The biochemical assay which consists of PH1˙-+ GOx has been further utilized as a glucose sensor. Upon addition of glucose (0-8 nM) in the biochemical assay, the in-situ produced H2O2 (after oxidation of glucose with GOx) oxidized PH1˙- to PH1 giving a sea green to pink colorimetric read out along with a decrease in the absorption intensities at 720, 815, 880 and 950 nm and the emergence of absorption intensity at 541 nm. The lowest limit of detection is 85 fM. We also explored this biochemical assay for the detection of 860 fM of glucose in a 10% blood serum. Similarly, fluorometric, CV and DPV studies were carried out for the detection of glucose using this biochemical assay. The smartphone-assisted RGB colour analyser showed large variations in the red colour and this RGB based colour differentiation can be used for the detection of 1 nM of glucose.

Local Regulation of Mitochondrial Respiratory for Enhancing Photodynamic Therapy and Breaking Chemoresistance in Hypoxia.

The hypoxic condition in solid tumors induces therapy resistance, limited therapeutic efficacy, and tumor recurrence, especially for chemotherapy and aerobic photodynamic therapy (PDT). To address this matter, an O2 regulator (SNP@Ato) is designed for breaking chemoresistance and enhancing PDT, which is constructed by loading Atovaquone (Ato) through self-assembly and host-guest interaction between β-cyclodextrin functionalized tetraphenylporphyrin (TPP-CD4) and thioketal-linked camptothecin/azobenzene (Azo-TK-CPT). Specifically, the porphyrin units in SNP@Ato are in "Off state" due to the photoinduced electron transfer (PET) effect between the porphyrin units and azobenzene. After encountering the hypoxic condition in solid tumors, SNP@Ato is dissociated by the cleaved azobenzene on account of over-expressed azo-reductase. Then the mitochondrial respiratory of cancer cells would be suppressed with the participation of Ato, generating a local hypoxia relief for sensitized chemotherapy and enhanced PDT. Accompanied by efficient PDT, the TK linker is broken by ROS, and the CPT is released from the prodrugs. Compared with the SNP group without oxygen-regulator, SNP@Ato exhibits a remarkable improvement of the therapeutic effect against hypoxic tumors in vitro and in vivo. This work proposes a novel paradigm for overcoming hypoxia-induced therapeutic resistance.

Characterization of MSC Growth, Differentiation, and EV Production in CNF Hydrogels Under Static and Dynamic Cultures in Hypoxic and Normoxic Conditions.

Mesenchymal stem cells (MSCs) hold immense therapeutic potential due to their regenerative and immunomodulatory properties. However, to utilize this potential, it is crucial to optimize their in vitro cultivation conditions. Three-dimensional (3D) culture methods using cell-laden hydrogels aim to mimic the physiological microenvironment in vitro, thus preserving MSC biological functionalities. Cellulosic hydrogels are particularly promising due to their biocompatibility, sustainability, and tunability in terms of chemical, morphological, and mechanical properties. This study investigated the impact of (1) two physical crosslinking scenarios for hydrogels derived from anionic cellulose nanofibers (to-CNF) used to encapsulate adipose-derived MSCs (adMSCs) and (2) physiological culture conditions on the in vitro proliferation, differentiation, and extracellular vesicle (EV) production of these adMSCs. The results revealed that additional Ca2+-mediated crosslinking, intended to complement the self-assembly and gelation of aqueous to-CNF in the adMSC cultivation medium, adversely affected both the mechanical properties of the hydrogel spheres and the growth of the encapsulated cells. However, cultivation under dynamic and hypoxic conditions significantly improved the proliferation and differentiation of the encapsulated adMSCs. Furthermore, it was demonstrated that the adMSCs in the CNF hydrogel spheres exhibited potential for scalable EV production with potent immunosuppressive capacities in a bioreactor system. These findings underscore the importance of physiological culture conditions and the suitability of cellulosic materials for enhancing the therapeutic potential of MSCs. Overall, this study provides valuable insights for optimizing the in vitro cultivation of MSCs for various applications, including tissue engineering, drug testing, and EV-based therapies.

A combination of covalent and noncovalent restricted‐intramolecular‐rotation strategy for supramolecular AIE‐type photosensitizer toward photodynamic therapy

AbstractPhotodynamic therapy (PDT) is a promising noninvasive method for targeted cancer cell destruction. Still, its effectiveness is often hindered by the aggregation‐caused quenching effect of organic photosensitizer (PS) in aqueous environments. Here, we have employed a combination of covalent and noncovalent restricted‐intramolecular‐rotation strategies to develop supramolecular PSs with aggregation‐induced emission (AIE) characteristics. Firstly, a water‐soluble octacationic molecular cage (1) with a bilayer tetraphenylethene (TPE) structure has been designed and synthesized, which minimizes intramolecular rotation of TPE moieties and achieves the single‐molecule‐level aggregation by the covalent restriction of intramolecular rotation (RIR) via molecular engineering synthesis. Compared with its single‐layer TPE analog, 1 exhibits superior efficiency in generating reactive oxygen species (ROS) including superoxide radical (O2−•) and singlet oxygen (1O2) upon white‐light irradiation. Subsequently, by forming a 1:4 host–guest complex (1@CB[8]4) between 1 and cucurbit[8]uril (CB[8]), O2−• generation can be further enhanced by the noncovalent RIR via the host–guest assembly. Additionally, 1@CB[8]4 as a photocatalyst promotes rapid oxidation of nicotinamide adenine dinucleotide (NADH) in water. Given its Type‐I ROS generation and catalytic activity for NADH oxidation, 1@CB[8]4 acts as a supramolecular AIE‐type PS to exhibit strong photo‐induced cytotoxicity upon white‐light irradiation under hypoxic conditions, showcasing its potential for synergistic PDT.

Proliferative Diabetic Retinopathy Microenvironment Drives Microglial Polarization and Promotes Angiogenesis and Fibrosis via Cyclooxygenase-2/Prostaglandin E2 Signaling

Diabetic retinopathy (DR) is the leading cause of visual impairment, particularly in the proliferative form (proliferative DR [PDR]). The impact of the PDR microenvironment on microglia, which are the resident immune cells in the central nervous system, and the specific pathological changes it may induce remain unclear. This study aimed to investigate the role of microglia in the progression of PDR under hypoxic and inflammatory conditions. We performed a comprehensive gene expression analysis using human-induced pluripotent stem cell-derived microglia under different stimuli (dimethyloxalylglycine (DMOG), lipopolysaccharide (LPS), and DMOG + LPS) to mimic the hypoxic inflammatory environment characteristic of PDR. Principal component analysis revealed distinct gene expression profiles, with 76 genes synergistically upregulated under combined stimulation. Notably, prostaglandin-endoperoxide synthase 2 (encoding cyclooxygenase (COX)-2) exhibited the most pronounced increase, leading to elevated prostaglandin E2 (PGE2) levels and driving pathological angiogenesis and inflammation via the COX-2/PGE2/PGE receptor 2 signaling axis. Additionally, the upregulation of the fibrogenic genes snail family transcriptional repressor 1 and collagen type I alpha 1 chain suggested a role for microglia in fibrosis. These findings underscore the critical involvement of microglia in PDR and suggest that targeting both the angiogenic and fibrotic pathways may present new therapeutic strategies for managing this condition.

Gas Tunnel Engineering of Prolyl Hydroxylase Reprograms Hypoxia Signaling in Cells

AbstractCells have evolved intricate mechanisms for recognizing and responding to changes in oxygen (O2) concentrations. Here, we have reprogrammed cellular hypoxia (low O2) signaling via gas tunnel engineering of prolyl hydroxylase 2 (PHD2), a non‐heme iron dependent O2 sensor. Using computational modeling and protein engineering techniques, we identify a gas tunnel and critical residues therein that limit the flow of O2 to PHD2’s catalytic core. We show that systematic modification of these residues can open the constriction topology of PHD2’s gas tunnel. Using kinetic stopped‐flow measurements with NO as a surrogate diatomic gas, we demonstrate up to 3.5‐fold enhancement in its association rate to the iron center of tunnel‐engineered mutants. Our most effectively designed mutant displays 9‐fold enhanced catalytic efficiency (kcat/KM=830±40 M−1 s−1) in hydroxylating a peptide mimic of hypoxia inducible transcription factor HIF‐1α, as compared to WT PHD2 (kcat/KM=90±9 M−1 s−1). Furthermore, transfection of plasmids that express designed PHD2 mutants in HEK‐293T mammalian cells reveal significant reduction of HIF‐1α and downstream hypoxia response transcripts under hypoxic conditions of 1 % O2. Overall, these studies highlight activation of PHD2 as a new pathway to reprogram hypoxia responses and HIF signaling in cells.

Screening of functional genes for hypoxia adaptation in Tibetan pigs by combined genome resequencing and transcriptome analysis.

The high-altitude, low-oxygen environment of the Qinghai-Tibet Plateau poses significant challenges for the introduction of superior livestock breeds. However, local plateau species have adapted to thrive and reproduce under these harsh conditions. Understanding the molecular mechanisms behind plateau animals' adaptation to low-oxygen environments is essential for breeding livestock suited to high-altitude regions. Tibetan pigs, which have undergone long-term natural selection and artificial breeding, have developed the ability to survive and reproduce in hypoxic environments. In this study, we conducted whole-genome resequencing of 30 Tibetan pigs from high-altitude regions and 30 Diannan small-ear pigs from low-altitude areas, to identify candidate genes that support Tibetan pigs' adaptation to hypoxic conditions through selection signal analysis. Additionally, we performed transcriptome sequencing on five tissues (heart, liver, spleen, lung, and bone marrow) from both Tibetan pigs and Diannan small-ear pigs to identify genes with significant differential expression between the two breeds. We then integrated the genomic and transcriptomic data by examining the expression of candidate genes identified in selection signal analysis across different tissues. The selection signal analysis identified 10 genes-HES4, ANGPT1, HIF3A, SPHK2, PCK2, RCN3, HIGD2A, DNM2, IRF9, and SRF-that were under positive selection in the Tibetan pig population and are associated with hypoxia adaptation. When combined with transcriptome data, we found that five of these genes-HIF3A, RCN3, HIGD2A, PCK2, and IRF9-exhibited differential expression. Through an integrated approach of selection signal and transcriptome analysis, we identified five key functional genes that contribute to the adaptation of Tibetan pigs to hypoxic environments. These findings offer new insights into the adaptability of plateau animals.

Delayed high-altitude retinal hemorrhages in well-acclimatized lowlanders: A hidden risk unveiled.

High-altitude retinal hemorrhage (HARH), a frequent entity of high-altitude retinopathy (HAR), is associated with acute exposure to high altitudes (HAs) with rapid ascent. However, the delayed occurrence of HARH in well-acclimatized individuals has received less attention. This study explored the prevalence and characteristics of HARH in individuals who were acclimatized to HA for extended periods, aiming to elucidate the physiological responses of the retina to chronic hypoxic conditions. We conducted an observational cross-sectional study involving 1,457 male participants who had resided at altitudes above 3353 meters (11,000 ft) in the Himalayan region for more than three months. Patients who had ocular or systemic diseases affecting the retina, chronic retinotoxic medication use or smoking were excluded. Participants underwent comprehensive ophthalmic examination, including the best corrected visual acuity test, dilated fundus examination and photography. A total of 1,457 males were screened, and the mean age was 34 ± 2 years. Of all the participants screened, 12 (0.82%) had HAR, with all cases occurring at altitudes above 4267 meters (14,000 ft). Seventy-five percent (75%) of the HAR patients had retinal hemorrhage and venous dilatation. Macular involvement and cotton wool spots were observed in 34% and 25% of HAR patients, respectively. Only four participants had foveal involvement with significant visual impairment. Delayed HARH in acclimatized individuals underscores the importance of long-term ocular monitoring for those exposed to HA, despite acclimatization, to prevent and manage potential visual impairments.

Gas Tunnel Engineering of Prolyl Hydroxylase Reprograms Hypoxia Signaling in Cells.

Cells have evolved intricate mechanisms for recognizing and responding to changes in oxygen (O2) concentrations. Here, we have reprogrammed cellular hypoxia (low O2) signaling via gas tunnel engineering of prolyl hydroxylase 2 (PHD2), a non-heme iron dependent O2 sensor. Using computational modeling and protein engineering techniques, we identify a gas tunnel and critical residues therein that limit the flow of O2 to PHD2's catalytic core. We show that systematic modification of these residues can open the constriction topology of PHD2's gas tunnel. Using kinetic stopped-flow measurements with NO as a surrogate diatomic gas, we demonstrate up to 3.5-fold enhancement in its association rate to the iron center of tunnel-engineered mutants. Our most effectively designed mutant displays 9-fold enhanced catalytic efficiency (kcat/KM=830±40 M-1 s-1) in hydroxylating a peptide mimic of hypoxia inducible transcription factor HIF-1α, as compared to WT PHD2 (kcat/KM=90±9 M-1 s-1). Furthermore, transfection of plasmids that express designed PHD2 mutants in HEK-293T mammalian cells reveal significant reduction of HIF-1α and downstream hypoxia response transcripts under hypoxic conditions of 1 % O2. Overall, these studies highlight activation of PHD2 as a new pathway to reprogram hypoxia responses and HIF signaling in cells.