Reprogramming the Tumor Immune Microenvironment Through Activatable Photothermal Therapy and GSH depletion Using Liposomal Gold Nanocages to Potentiate Anti-Metastatic Immunotherapy.

Cancer immunotherapy offers significant clinical benefits for patients with advanced or metastatic tumors. However, immunotherapeutic efficacy is often hindered by the tumor microenvironment's high redox levels, leading to variable patient outcomes. Herein, a therapeutic liposomal gold nanocage (MGL) is innovatively developed based on photo-triggered hyperthermia and a releasable strategy by combining a glutathione (GSH) depletion to remodel the tumor immune microenvironment, fostering a more robust anti-tumor immune response. MGL comprises a thermosensitive liposome shell and a gold nanocage core loaded with maleimide. The flexible shell promotes efficient uptake by cancer cells, enabling targeted destruction through photothermal therapy while triggering immunogenic cell death and the maturation of antigen-presenting cells. The photoactivated release of maleimide depletes intracellular GSH, increasing tumor cell sensitivity to oxidative stress and thermal damage. Conversely, GSH reduction also diminishes immunosuppressive cell activity, enhances antigen presentation, and activates T cells. Moreover, photothermal immunotherapy decreases elevated levels of heat shock proteins in tumor cells, further increasing their sensitivity to hyperthermia. In summary, MGL elicited a robust systemic antitumor immune response through GSH depletion, facilitating an effective photothermal immunotherapeutic strategy that reprograms the tumor microenvironment and significantly inhibits primary and metastatic tumors. This approach demonstrates considerable translational potential and clinical applicability.

COVID-19 Vaccine mRNA Biodistribution: Maternal and Fetal Exposure Risks.

SARS-CoV-2 infection during pregnancy has severe consequences on maternal and neonatal health. Presently, vaccination stands as a critical preventive measure for mitigating infection-related risks. Although the initial clinical trials for the COVID-19 vaccines excluded pregnant women, subsequent investigations have indicated mRNA vaccinations' effectiveness and short-term safety during pregnancy. However, there is a lack of information regarding the potential biodistribution of the vaccine mRNA during pregnancy and lactation. Recent findings indicate that COVID-19 vaccine mRNA has been detected in breast milk, suggesting that its presence is not confined to the injection site and raises the possibility of similar distribution to the placenta and the fetus. Furthermore, the potential effects and responses of the placenta and fetus to the vaccine mRNA are still unknown. While potential risks might exist with the exposure of the placenta and fetus to the COVID-19 mRNA vaccine, the application of mRNA therapies for maternal and fetal conditions offers a groundbreaking prospect. Future research should leverage the unique opportunity provided by the first-ever application of mRNA vaccines in humans to understand their biodistribution and impact on the placenta and fetus in pregnant women. Such insights could substantially advance the development of safer and more effective future mRNA-based therapies during pregnancy.

Overlapping peri-implantation phenotypes of ZNHIT1 and ZNHIT2 despite distinct functions during early mouse development.

Mammalian preimplantation development culminates in the formation of a blastocyst which undergoes extensive gene expression regulation to successfully implant into the maternal endometrium. Zinc-finger HIT domain-containing (ZNHIT) 1 and 2 are members of a highly conserved family, yet they have been identified as subunits of distinct complexes. Here we report that knockout of either Znhit1 or Znhit2 results in embryonic lethality during peri-implantation stages. Znhit1 and Znhit2 mutant embryos have overlapping phenotypes, including reduced proportion of SOX2-positive ICM cells, a lack of Fgf4 expression and aberrant expression of NANOG and SOX17. Furthermore, we find that the similar phenotypes are caused by distinct mechanisms. Specifically, embryos lacking ZNHIT1 likely fail to incorporate sufficient H2A.Z at the promoter region of Fgf4 and other genes involved in cell projection organization resulting in impaired invasion of trophoblast cells during implantation. In contrast, Znhit2 mutant embryos display a complete lack of nuclear EFTUD2, a key component of U5 spliceosome, indicating a global splicing deficiency. Our findings unveil the indispensable yet distinct roles of ZNHIT1 and ZNHIT2 in early mammalian embryonic development.

Design and Application of pH-Responsive Liposomes for Site-Specific Delivery of Cytotoxin from Cobra Venom.

Background: Current immunotherapies with unexpected severe side effects and treatment resistance have not resulted in the desired outcomes for patients with melanoma, and there is a need to discover more effective medications. Cytotoxin (CTX) from Cobra Venom has been established to have favorable cytolytic activity and antitumor efficacy and is regarded as a promising novel anticancer agent. However, amphiphilic CTX with excellent anionic phosphatidylserine lipid-binding ability may also damage normal cells. Methods: We developed pH-responsive liposomes with a high CTX load (CTX@PSL) for targeted acidic-stimuli release of drugs in the tumor microenvironment. The morphology, size, zeta potential, drug-release kinetics, and preservation stability were characterized. Cell uptake, apoptosis-promoting effects, and cytotoxicity were assessed using MTT assay and flow cytometry. Finally, the tissue distribution and antitumor effects of CTX@PSL were systematically assessed using an in vivo imaging system. Results: CTX@PSL exhibited high drug entrapment efficiency, drug loading, stability, and a rapid release profile under acidic conditions. These nanoparticles, irregularly spherical in shape and small in size, can effectively accumulate at tumor sites (six times higher than free CTX) and are rapidly internalized into cancer cells (2.5-fold higher cell uptake efficiency). CTX@PSL displayed significantly stronger cytotoxicity (IC50 0.25 µg/mL) and increased apoptosis in than the other formulations (apoptosis rate 71.78±1.70%). CTX@PSL showed considerably better tumor inhibition efficacy than free CTX or conventional liposomes (tumor inhibition rate 79.78±5.93%). Conclusion: Our results suggest that CTX@PSL improves tumor-site accumulation and intracellular uptake for sustained and targeted CTX release. By combining the advantages of CTX and stimuli-responsive nanotechnology, the novel CTX@PSL nanoformulation is a promising therapeutic candidate for cancer treatment.

A homogeneous electrochemiluminescence immunoassay platform based on carbon quantum dots and magnetic beads enrichment for detection of thyroglobulin in serum.

Considering the high probability of recurrence or metastasis after thyroidectomy, it is meaningful to develop a rapid, sensitive and specific method for monitoring thyrophyma-related biomarkers. In this study, a homogeneous electrochemiluminescence immunoassay (HO-ECLIA) coupled with magnetic beads (MBs)-based enrichment tactic was established for the determination of thyrophyma-related thyroglobulin (Tg). Importantly, owing to the abundant surface groups and good biocompatibility of carbon quantum dots (CQDs), the incorporation of CQDs onto the Tg antigen surface was achieved, resulting in the formation of Tg-encapsulated CQDs (CQDs-Tg), which served not only as an ECL probe but as a biorecognition element. Under optimal experimental conditions, the proposed platform demonstrated a wide linear range from 0.01 to 100 ng·mL-1 with a detection limit of 6.9 pg·mL-1 (S/N = 3), and performed well in real serum sample analysis against interference. Collectively, the proposed platform exhibited the rapid response, satisfactory sensitivity and specificity toward Tg in complex serum milieu, and held a considerable potential for clinical prognosis monitoring of thyrophyma.

Cervical extracellular matrix hydrogel optimizes tumor heterogeneity of cervical squamous cell carcinoma organoids.

Cervical cancer, primarily squamous cell carcinoma, is the most prevalent gynecologic malignancy. Organoids can mimic tumor development in vitro, but current Matrigel inaccurately replicates the tissue-specific microenvironment. This limitation compromises the accurate representation of tumor heterogeneity. We collected para-cancerous cervical tissues from patients diagnosed with cervical squamous cell carcinoma (CSCC) and prepared uterine cervix extracellular matrix (UCEM) hydrogels. Proteomic analysis of UCEM identified several tissue-specific signaling pathways including human papillomavirus, phosphatidylinositol 3-kinase-AKT, and extracellular matrix receptor. Secreted proteins like FLNA, MYH9, HSPA8, and EEF1A1 were present, indicating UCEM successfully maintained cervical proteins. UCEM provided a tailored microenvironment for CSCC organoids, enabling formation and growth while preserving tumorigenic potential. RNA sequencing showed UCEM-organoids exhibited greater similarity to native CSCC and reflected tumor heterogeneity by exhibiting CSCC-associated signaling pathways including virus protein-cytokine, nuclear factor κB, tumor necrosis factor, and oncogenes EGR1, FPR1, and IFI6. Moreover, UCEM-organoids developed chemotherapy resistance. Our research provides insights into advanced organoid technology through native matrix hydrogels.

Corrigendum to "Anti-tumor effect and mechanism of the total biflavonoid extract from S doederleinii on human cervical cancer cells in vitro and in vivo" [Heliyon 10(2) January 2024 e24778].

[This corrects the article DOI: 10.1016/j.heliyon.2024.e24778.].

Vertical transmission of SARS-CoV-2 delta-variant in a preterm infant.

BACKGROUND: As SARS-CoV-2 continues to be relevant and cause illnesses, the effect of emerging virus variants on perinatal health remains to be elucidated. It was demonstrated that vertical transmission of SARS-CoV-2 is a relatively rare event in the original SARS-CoV-2 strain. However, very few reports describe vertical transmission related to the delta-variant. CASE PRESENTATION: We report a case of a preterm male neonate born to a mother with positive SARS-CoV-2 and mild respiratory complications. The neonate was born by cesarean section due to fetal distress. The rupture of the amniotic membrane was at delivery. The neonate had expected prematurity-related complications. His nasopharyngeal swabs for RT-PCR were positive from birth till three weeks of age. RT-ddPCR of the Placenta showed a high load of the SARS-CoV-2 virus with subgenomic viral RNA. RNAscope technique demonstrated both the positive strand of the S gene and the orf1ab negative strand. Detection of subgenomic RNA and the orf1ab negative strand indicats active viral replication in the placenta. CONCLUSIONS: Our report demonstrates active viral replication of the SARS-CoV-2 delta-variant in the placenta associated with vertical transmission in a preterm infant.

Elucidating the role of the GC/GR/GLUT1 axis in steroid-induced osteonecrosis of the femoral head: A proteomic approach.

BACKGROUND: Steroid-induced osteonecrosis of the femoral head (SONFH) is a prevalent and incapacitating condition that affects the hip joint. Unfortunately, early diagnostic and treatment measures are limited. METHODS: Our study employed Tandem Mass Tag (TMT) labeling mass spectrometry (MS)-based quantitative proteome to compare the proteins of femoral head tissues in patients with SONFH with those of patients who sustained femoral neck fracture (FNF). We investigated the level and effects of glucose transporter member 1 (GLUT1) in SONFH patients and MC3T3-E1 cells and examined the function and molecular mechanism of GLUT1 in the context of SONFH using in vivo and in vitro approaches. RESULTS: The SONFH group exhibited significant changes in protein expression levels compared to the fracture group. Specifically, we observed the up-regulation of 86 proteins and the down-regulation of 138 proteins in the SONFH group. Among the differentially expressed proteins, GLUT1 was down-regulated and associated with glucose metabolic processes in the SONFH group. Further analysis using Parallel Reaction Monitoring (PRM), WB, and PCR confirmed that the protein was significantly down-regulated in both femoral head tissue samples from SONFH patients and dexamethasone-treated MC3T3-E1 cells. Moreover, overexpression of GLUT1 effectively reduced glucocorticoid (GC)-induced apoptosis and the suppression of osteoblast proliferation and osteogenic differentiation in MC3T3-E1 cells, as well as GC-induced femoral head destruction in GC-induced ONFH rat models. Additionally, our research demonstrated that GC down-regulated GLUT1 transcription via glucocorticoid receptors in MC3T3-E1 cells. CONCLUSIONS: GLUT1 was down-regulated in patients with SONFH; furthermore, down-regulated GLUT1 promoted apoptosis and inhibited osteoblast ossification in dexamethasone-induced MC3T3-E1 cells and contributed to GC-induced femoral head destruction in a SONFH rat model. Glucocorticoids inhibited the transcriptional activity of GLUT1, leading to a reduction in the amount and activity of GLUT1 in the cells and ultimately promoting apoptosis and inhibiting osteoblast ossification via the GC/GR/GLUT1 axis in SONFH.

Establishment of a human organoid-based evaluation system for assessing interspecies infection risk of animal-borne coronaviruses.

The COVID-19 pandemic presents a major threat to global public health. Several lines of evidence have shown that the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), along with two other highly pathogenic coronaviruses, SARS-CoV and Middle East Respiratory Syndrome (MERS-CoV) originated from bats. To prevent and control future coronavirus outbreaks, it is necessary to investigate the interspecies infection and pathogenicity risks of animal-related coronaviruses. Currently used infection models, including in vitro cell lines and in vivo animal models, fail to fully mimic the primary infection in human tissues. Here, we employed organoid technology as a promising new model for studying emerging pathogens and their pathogenic mechanisms. We investigated the key host-virus interaction patterns of five human coronaviruses (SARS-CoV-2 original strain, Omicron BA.1, MERS-CoV, HCoV-229E, and HCoV-OC43) in different human respiratory organoids. Five indicators, including cell tropism, invasion preference, replication activity, host response and virus-induced cell death, were developed to establish a comprehensive evaluation system to predict coronavirus interspecies infection and pathogenicity risks. Using this system, we further examined the pathogenicity and interspecies infection risks of three SARS-related coronaviruses (SARSr-CoV), including WIV1 and rRsSHC014S from bats, and MpCoV-GX from pangolins. Moreover, we found that cannabidiol, a non-psychoactive plant extract, exhibits significant inhibitory effects on various coronaviruses in human lung organoid. Cannabidiol significantly enhanced interferon-stimulated gene expression but reduced levels of inflammatory cytokines. In summary, our study established a reliable comprehensive evaluation system to analyse infection and pathogenicity patterns of zoonotic coronaviruses, which could aid in prevention and control of potentially emerging coronavirus diseases.

Clinical Analysis of Atorvastatin Calcium, Fenofibrate, and Acipimox in the Treatment of Hypertriglyceridemia-induced Acute Pancreatitis.

Background: Hypertriglyceridemia-induced acute pancreatitis (HTG-AP) is an increasingly recognized and potentially severe form of acute pancreatitis. The effective management of HTG-AP is critical due to its association with significant morbidity and mortality. HTG-AP poses a considerable burden on affected individuals and healthcare systems. It can result in persistent upper abdominal pain, nausea, vomiting, abdominal distension, fever, and in severe cases, hypotension or shock and multiple organ dysfunction. Standard treatment strategies often involve lipid-lowering agents, but the optimal therapeutic approach remains a subject of ongoing research. This study aims to evaluate the efficacy of atorvastatin calcium, fenofibrate, and acipimox, either individually or in combination, in the treatment of HTG-AP, providing insights into more effective management strategies. Methods: 150 HTG-AP patients admitted to the first hospital of Putian from June 2020 to December 2022 were selected. The age range of the patients included in the study was between 30 and 70 years, with an average age of approximately 48 years. The cohort consisted of 90 males and 60 females, resulting in a male-to-female ratio of 3:2. The patients were grouped: atorvastatin calcium, acipimox, fenofibrate, fenofibrate + Atorvastatin calcium, fenofibrate + acipimox, and no drug. The therapeutic effects and clinical indicators of the six groups were compared. Results: Patients in the fenofibrate + acipimox and fenofibrate groups experienced significantly reduced hospitalization duration compared to the other groups. They also had shorter abdominal pain relief time and gastrointestinal function relief time. Additionally, these groups had lower peak levels of amylase (an enzyme) and cholesterol compared to the other groups. In terms of neutrophil (NEUT) increase, the fenofibrate + acipimox, atorvastatin calcium, and fenofibrate groups had significantly lower peak levels compared to the other groups, indicating a less pronounced increase in NEUT. Furthermore, the fenofibrate and acipimox groups exhibited significantly lower peak levels of C-reactive protein (CRP) compared to the other groups. CRP is an indicator of inflammation. On the other hand, the atorvastatin calcium group had higher levels of procalcitonin (a marker of infection) and a higher peak score on the acute physiology and chronic health evaluation II (APACHE II) scale, which assesses the severity of acute pancreatitis, compared to the other groups (all P < .05). Conclusion: The findings of this study highlight the effectiveness of combining fenofibrate and acipimox in the treatment of HTG-AP, leading to rapid disease recovery and significant improvement in clinical symptoms. These results have important implications for clinical practice, as the combination therapy can be widely adopted as an effective treatment strategy for HTG-AP patients. Moreover, this study provides valuable insights into the management of HTG-AP and suggests that lipid-lowering agents, such as atorvastatin calcium and fenofibrate, play a crucial role in the treatment of this condition. However, further research is needed to explore the optimal dosages, treatment durations, and potential side effects of these medications in HTG-AP patients.

Anti-tumor effect and mechanism of the total biflavonoid extract from S doederleinii on human cervical cancer cells in vitro and in vivo.

In this study, the therapeutic effect and possible mechanism of the total biflavonoid extract of Selaginella doederleinii Hieron (SDTBE) against cervical cancer were originally investigated in vitro and in vivo. First, the inhibition of SDTBE on proliferation of cervical cancer HeLa cells was evaluated, followed by morphological observation with AO/EB staining, Annexin V/PI assay, and autophagic flux monitoring to evaluate the possible effect of SDTBE on cell apoptosis and autophagy. Cell cycle, as well as mitochondrial membrane potential (ΔÑ°m), was detected with flow cytometry. Further, the apoptosis related protein expression and the autophagy related gene LC3 mRNA transcription level were analyzed by Western blot (WB) and real-time quantitative polymerase chain reaction (RT-qPCR), respectively. Finally, the anti-cervical cancer effect of the SDTBE was also validated in vivo in HeLa cells grafts mice. As results, SDTBE inhibited HeLa cells proliferation with the IC50 values of 49.05 ± 6.76 and 44.14 ± 4.75 µg/mL for 48 and 72 h treatment, respectively. The extract caused mitochondrial ΔÑ° loss, induced cell apoptosis by upregulating Bax, downregulating Bcl-2, activating Caspase-9 and Caspase-3, promoting cell autophagy and blocking the cell cycle in G0/G1 phase. Furthermore, 100, 200, and 300 mg/kg SDTBE suppressed the growth of HeLa cells xenografts in mice with the mean inhibition rates, 25.3 %, 57.5 % and 62.9 %, respectively, and the change of apoptosis related proteins and microvascular density was confirmed in xenografts by immunohistochemistry analysis. The results show that SDTBE possesses anti-cervical cancer effect, and the mechanism involves in activating Caspase-dependent mitochondrial apoptosis pathway.

Simultaneous antioxidant and neuroprotective effects of two-dimensional (2D) MXene-loaded isoquercetin for ischemic stroke treatment.

Oxidative stress and reactive oxygen species drive ischemic stroke and its related complications. New antioxidant medications are therefore crucial for treating ischemic stroke. We developed Ti2C@BSA-ISO nanocomposites loaded with the hydrophobic drug isoquercetin (ISO) encapsulated in BSA on Ti2C nano-enzymes as a novel therapeutic nanomedicine for the treatment of ischemic stroke targeting reactive oxygen species (ROS). TEM visually proved the successful preparation of Ti2C@BSA-ISO, and the FTIR, XPS, zeta potential and DLS together demonstrated the acquisition of Ti2C@BSA-ISO. In addition, the enzyme-mimicking activity of Ti2C was evaluated and the antioxidant capacity of Ti2C@BSA-ISO was verified. Ti2C@BSA-ISO was able to reverse the decrease in cellular activity caused by ROS. Experiments in vivo showed that Ti2C@BSA-ISO could promote neuroprotection and scavenging of ROS in the hippocampal CA1 area and cerebral cortex of rats, thereby inhibiting cellular death and alleviating ischaemic stroke. Specifically, Ti2C@BSA-ISO alleviated ischemic stroke by inhibiting NLRP3/caspase-1/GSDMD pathway-mediated pyroptosis. Our study demonstrates the effectiveness of nanomedicines that can be directly used as drugs for the treatment of ischemic stroke in synergy with other drugs, which greatly expands the application of nanomaterials in the treatment of ischemic stroke.

Immunoactivity of a hybrid membrane biosurface on nanoparticles: enhancing interactions with dendritic cells to augment anti-tumor immune responses.

Nano-biointerfaces play a pivotal role in determining the functionality of engineered therapeutic nanoparticles, particularly in the context of designing nanovaccines to effectively activate immune cells for cancer immunotherapy. Unlike involving chemical reactions by conventional surface decorating strategies, cell membrane-coating technology offers a straightforward approach to endow nanoparticles with natural biosurfaces, enabling them to mimic and integrate into the intricate biosystems of the body to interact with specific cells under physiological conditions. In this study, cell membranes, in a hybrid formulation, derived from cancer and activated macrophage cells were found to enhance the interaction of nanoparticles (HMP) with dendritic cells (DCs) and T cells among the mixed immune cells from lymph nodes (LNs), which could be leveraged in the development of nanovaccines for anti-tumor therapy. After loading with an adjuvant (R837), the nanoparticles coated with a hybrid membrane (HMPR) demonstrated effectiveness in priming DCs both in vitro and in vivo, resulting in amplified anti-tumor immune responses compared to those of nanoparticles coated with a single type of membrane or those lacking a membrane coating. The elevated immunoactivity of nanoparticles achieved by incorporating a hybrid membrane biosurface provides us a more profound comprehension of the nano-immune interaction, which may significantly benefit the development of bioactive nanomaterials for advanced therapy.

Transcriptional regulation of FACT involves Coordination of chromatin accessibility and CTCF binding.

Histone chaperone FACT (facilitates chromatin transcription) is well known to promote chromatin recovery during transcription. However, the mechanism how FACT regulates genome-wide chromatin accessibility and transcription factor binding has not been fully elucidated. Through loss-of-function studies, we show here that FACT component Ssrp1 is required for DNA replication and DNA damage repair and is also essential for progression of cell phase transition and cell proliferation in mouse embryonic fibroblast cells. On the molecular level, absence of the Ssrp1 leads to increased chromatin accessibility, enhanced CTCF binding, and a remarkable change in dynamic range of gene expression. Our study thus unequivocally uncovers a unique mechanism by which FACT complex regulates transcription by coordinating genome-wide chromatin accessibility and CTCF binding.

Micelle-mediated chemiluminescence of 6-aza-2-thiothymine-protected gold nanoclusters for carbazochrome sodium sulfonate detection.

Chemiluminescence (CL) intensity of luminol-H2O2 system was dramatically enhanced by cetyltrimethylammonium bromide (CTAB) micelle-mediated 6-aza-2-thiothymine-protected gold nanoclusters (ATT-AuNCs). It is proved that spherical micelles of CTAB in aqueous solution improved the dispersity of ATT-AuNCs, thus enhancing their catalytic activity, which brought in the increased CL intensity of luminol-H2O2 system. Carbazochrome sodium sulfonate (CSS) with a hemostatic containing tetrahydroindole structure broke the spherical micelles and notably quenched the CL intensity of luminol-H2O2-CTAB-ATT AuNCs system. Based on these results, a simple, fast, and sensitive CL method has been developed for the detection of CSS with a linear range of 0.25-25 µM and a detection limit of 0.11 µM. The method has also been successfully applied to the determination of CSS in serum with satisfied recoveries in the range of 89.6 % to 103.7 %. This study not only provides an effective approach for CSS detection but also paves the way for AuNCs-based CL applications.

The unique function of Runx1 in skeletal muscle differentiation and regeneration is mediated by an ETS interaction domain.

The conserved Runt-related (RUNX) transcription factor family are well-known master regulators of developmental and regenerative processes. Runx1 and Runx2 are both expressed in satellite cells (SC) and skeletal myotubes. Conditional deletion of Runx1 in adult SC negatively impacted self-renewal and impaired skeletal muscle maintenance. Runx1- deficient SC retain Runx2 expression but cannot support muscle regeneration in response to injury. To determine the unique molecular functions of Runx1 that cannot be compensated by Runx2 we deleted Runx1 in C2C12 that retain Runx2 expression and established that myoblasts differentiation was blocked in vitro due in part to ectopic expression of Mef2c, a target repressed by Runx1 . Structure-function analysis demonstrated that the Ets-interacting MID/EID region of Runx1, absent from Runx2, is critical to regulating myoblasts proliferation, differentiation, and fusion. Analysis of in-house and published ChIP-seq datasets from Runx1 (T-cells, muscle) versus Runx2 (preosteoblasts) dependent tissue identified enrichment for a Ets:Runx composite site in Runx1 -dependent tissues. Comparing ATACseq datasets from WT and Runx1KO C2C12 cells showed that the Ets:Runx composite motif was enriched in peaks open exclusively in WT cells compared to peaks unique to Runx1KO cells. Thus, engagement of a set of targets by the RUNX1/ETS complex define the non-redundant functions of Runx1 .