Development and validation of prechiasmatic mouse model of subarachnoid hemorrhage to measure long-term neurobehavioral impairment.

Controllable and reproducible animal models of aneurysmal subarachnoid hemorrhage (SAH) are crucial for the systematic study of the pathophysiology and treatment of this debilitating condition. Despite the variety of animal models of SAH currently available, attempts to translate promising therapeutic strategies from preclinical studies to humans have largely failed. This failure is likely due, at least in part, to poor replication of pathology and disabilities in these preclinical models, especially the long-term neurocognitive deficits that drive poor quality of life / return to work in SAH survivors. Therefore, there is an unmet need to develop experimental models that reliably replicate the long-term clinical ramifications of SAH - especially in mice where genetic manipulations are straightforward and readily available. To address this need, we developed a standardized mouse model of SAH that reproducibly produced significant and trackable long-term neurobehavioral deficits. SAH was induced by performing double blood injections into the prechiasmatic cistern - a simple modification to the well-characterized single prechiasmatic injection mouse model of SAH. Following SAH, mice recapitulated key characteristics of SAH patients including long-term cognitive impairment as observed by a battery of behavioral testing and delayed pathophysiologic processes assayed by neuroinflammatory markers. We believe that this new SAH mouse model will be an ideal paradigm for investigating the complex pathophysiology of SAH and identifying novel druggable therapeutic targets for treating SAH-associated long-term neurocognitive deficits in patients.

Propofol Affords No Protection against Delayed Cerebral Ischemia in a Mouse Model of Subarachnoid Hemorrhage.

Delayed cerebral ischemia (DCI) is an important contributor to poor outcomes in aneurysmal subarachnoid hemorrhage (SAH) patients. We previously showed that volatile anesthetics such as isoflurane, sevoflurane and desflurane provided robust protection against SAH-induced DCI, but the impact of a more commonly used intravenous anesthetic agent, propofol, is not known. The goal of our current study is to examine the neurovascular protective effects of propofol on SAH-induced DCI. Twelve-week-old male wild-type mice were utilized for the study. Mice underwent endovascular perforation SAH or sham surgery followed one hour later by propofol infusion through the internal jugular vein (2 mg/kg/min continuous intravenous infusion). Large artery vasospasm was assessed three days after SAH. Neurological outcome assessment was performed at baseline and then daily until animal sacrifice. Statistical analysis was performed via one-way ANOVA and two-way repeated measures ANOVA followed by the Newman-Keuls multiple comparison test with significance set at p < 0.05. Intravenous propofol did not provide any protection against large artery vasospasm or sensory-motor neurological deficits induced by SAH. Our data show that propofol did not afford significant protection against SAH-induced DCI. These results are consistent with recent clinical studies that suggest that the neurovascular protection afforded by anesthetic conditioning is critically dependent on the class of anesthetic agent.

Isoflurane Conditioning-Induced Delayed Cerebral Ischemia Protection in Subarachnoid Hemorrhage-Role of Inducible Nitric Oxide Synthase.

Background Recent evidence implicates inflammation as a key driver in delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage (SAH). Inducible nitric oxide synthase (iNOS) is one of the known major mediators of inflammation. We previously showed that an inhalational anesthetic, isoflurane, provides strong protection against delayed cerebral ischemia after SAH. Our current study aims to define the role of iNOS in isoflurane conditioning-induced protection against delayed cerebral ischemia in a mouse model of SAH. Methods and Results The experiments used 10- to 14-week-old male wild-type (C57BL/6) and iNOS global knockout mice. Anesthetic conditioning was initiated 1 hour after SAH with isoflurane 2% for 1 hour. Isoflurane-induced changes in iNOS expression were measured. N-(3-(aminomethyl) benzyl) acetamidine, a highly selective iNOS inhibitor, was injected intraperitoneally immediately after SAH and then daily. Vasospasm, microvessel thrombosis, and neurological assessment was performed. Data were analyzed by 1-way ANOVA and 2-way repeated measures ANOVA followed by Student Newman Keuls comparison test. Statistical significance was set at P<0.05. Isoflurane conditioning downregulated iNOS expression in naïve and SAH mice. N-(3-(aminomethyl) benzyl) acetamidine attenuated large artery vasospasm and microvessel thrombosis and improved neurological deficits in wild-type animals. iNOS knockout mice were significantly resistant to vasospasm, microvessel thrombosis, and neurological deficits induced by SAH. Combining isoflurane with N-(3-(aminomethyl) benzyl) acetamidine did not offer extra protection, nor did treating iNOS knockout mice with isoflurane. Conclusions Isoflurane conditioning-induced delayed cerebral ischemia protection appears to be mediated by downregulating iNOS. iNOS is a potential therapeutic target to improve outcomes after SAH.

Isoflurane Conditioning Provides Protection against Subarachnoid Hemorrhage Induced Delayed Cerebral Ischemia through NF-kB Inhibition.

Delayed cerebral ischemia (DCI) is the largest treatable cause of poor outcome after aneurysmal subarachnoid hemorrhage (SAH). Nuclear Factor Kappa-light-chain-enhancer of Activated B cells (NF-kB), a transcription factor known to function as a pivotal mediator of inflammation, is upregulated in SAH and is pathologically associated with vasospasm. We previously showed that a brief exposure to isoflurane, an inhalational anesthetic, provided multifaceted protection against DCI after SAH. The aim of our current study is to investigate the role of NF-kB in isoflurane-conditioning-induced neurovascular protection against SAH-induced DCI. Twelve-week-old wild type male mice (C57BL/6) were divided into five groups: sham, SAH, SAH + Pyrrolidine dithiocarbamate (PDTC, a selective NF-kB inhibitor), SAH + isoflurane conditioning, and SAH + PDTC with isoflurane conditioning. Experimental SAH was performed via endovascular perforation. Anesthetic conditioning was performed with isoflurane 2% for 1 h, 1 h after SAH. Three doses of PDTC (100 mg/kg) were injected intraperitoneally. NF-kB and microglial activation and the cellular source of NF-kB after SAH were assessed by immunofluorescence staining. Vasospasm, microvessel thrombosis, and neuroscore were assessed. NF-kB was activated after SAH; it was attenuated by isoflurane conditioning. Microglia was activated and found to be a major source of NF-kB expression after SAH. Isoflurane conditioning attenuated microglial activation and NF-kB expression in microglia after SAH. Isoflurane conditioning and PDTC individually attenuated large artery vasospasm and microvessel thrombosis, leading to improved neurological deficits after SAH. The addition of isoflurane to the PDTC group did not provide any additional DCI protection. These data indicate isoflurane-conditioning-induced DCI protection after SAH is mediated, at least in part, via downregulating the NF-kB pathway.

Alterations of the gut microbial community structure and function with aging in the spontaneously hypertensive stroke prone rat.

Gut dysbiosis, a pathological imbalance of bacteria, has been shown to contribute to the development of hypertension (HT), systemic- and neuro-inflammation, and blood-brain barrier (BBB) disruption in spontaneously hypertensive stroke prone rats (SHRSP). However, to date individual species that contribute to HT in the SHRSP model have not been identified. One potential reason, is that nearly all studies of the SHRSP gut microbiota have analyzed samples from rats with established HT. The goal of this study was to examine the SHRSP gut microbiota before, during, and after the onset of hypertension, and in normotensive WKY control rats over the same age range. We hypothesized that we could identify key microbes involved in the development of HT by comparing WKY and SHRSP microbiota during the pre-hypertensive state and longitudinally. Systolic blood pressure (SBP) was measured by tail-cuff plethysmography and fecal microbiota analyzed by16S rRNA gene sequencing. SHRSP showed significant elevations in SBP, as compared to WKY, beginning at 8 weeks of age (p < 0.05 at each time point). Bacterial community structure was significantly different between WKY and SHRSP as early as 4 weeks of age, and remained different throughout the study (p = 0.001-0.01). At the phylum level we observed significantly reduced Firmicutes and Deferribacterota, and elevated Bacteroidota, Verrucomicrobiota, and Proteobacteria, in pre-hypertensive SHRSP, as compared to WKY. At the genus level we identified 18 bacteria whose relative abundance was significantly different in SHRSP versus WKY at the pre-hypertensive ages of 4 or 6 weeks. In an attempt to further refine bacterial candidates that might contribute to the SHRSP phenotype, we compared the functional capacity of WKY versus SHRSP microbial communities. We identified significant differences in amino acid metabolism. Using untargeted metabolomics we found significant reductions in metabolites of the tryptophan-kynurenine pathway and increased indole metabolites in SHRSP versus WKY plasma. Overall, we provide further evidence that gut dysbiosis contributes to hypertension in the SHRSP model, and suggest for the first time the potential involvement of tryptophan metabolizing microbes.

Engineered pegRNAs improve prime editing efficiency.

Prime editing enables the installation of virtually any combination of point mutations, small insertions or small deletions in the DNA of living cells. A prime editing guide RNA (pegRNA) directs the prime editor protein to the targeted locus and also encodes the desired edit. Here we show that degradation of the 3' region of the pegRNA that contains the reverse transcriptase template and the primer binding site can poison the activity of prime editing systems, impeding editing efficiency. We incorporated structured RNA motifs to the 3' terminus of pegRNAs that enhance their stability and prevent degradation of the 3' extension. The resulting engineered pegRNAs (epegRNAs) improve prime editing efficiency 3-4-fold in HeLa, U2OS and K562 cells and in primary human fibroblasts without increasing off-target editing activity. We optimized the choice of 3' structural motif and developed pegLIT, a computational tool to identify non-interfering nucleotide linkers between pegRNAs and 3' motifs. Finally, we showed that epegRNAs enhance the efficiency of the installation or correction of disease-relevant mutations.

STAT3 inhibitor mitigates cerebral amyloid angiopathy and parenchymal amyloid plaques while improving cognitive functions and brain networks.

Previous reports indicate a potential role for signal transducer and activator of transcription 3 (STAT3) in amyloid-ß (Aß) processing and neuritic plaque pathogenesis. In the present study, the impact of STAT3 inhibition on cognition, cerebrovascular function, amyloid pathology, oxidative stress, and neuroinflammation was studied using in vitro and in vivo models of Alzheimer's disease (AD)-related pathology. For in vitro experiments, human brain vascular smooth muscle cells (HBVSMC) and human brain microvascular endothelial cells (HBMEC) were used, and these cultured cells were exposed to Aß peptides followed by measurement of activated forms of STAT3 expression and reactive oxygen species (ROS) generation. Further, 6 months old 5XFAD/APOE4 (5XE4) mice and age-matched negative littermates were used for in vivo experiments. These mice were treated with STAT3 specific inhibitor, LLL-12 for 2 months followed by neurobehavioral and histopathological assessment. In vitro experiments showed exposure of cerebrovascular cells to Aß peptides upregulated activated forms of STAT3 and produced STAT3-mediated vascular oxidative stress. 5XE4 mice treated with the STAT3-specific inhibitor (LLL-12) improved cognitive functions and functional connectivity and augmented cerebral blood flow. These functional improvements were associated with a reduction in neuritic plaques, cerebral amyloid angiopathy (CAA), oxidative stress, and neuroinflammation. Reduction in amyloid precursor protein (APP) processing and attenuation of oxidative modification of lipoprotein receptor related protein-1 (LRP-1) were identified as potential underlying mechanisms. These results demonstrate the broad impact of STAT3 on cognitive functions, parenchymal and vascular amyloid pathology and highlight the therapeutic potential of STAT3 specific inhibition for treatment of AD and CAA.

Enhanced prime editing systems by manipulating cellular determinants of editing outcomes.

While prime editing enables precise sequence changes in DNA, cellular determinants of prime editing remain poorly understood. Using pooled CRISPRi screens, we discovered that DNA mismatch repair (MMR) impedes prime editing and promotes undesired indel byproducts. We developed PE4 and PE5 prime editing systems in which transient expression of an engineered MMR-inhibiting protein enhances the efficiency of substitution, small insertion, and small deletion prime edits by an average 7.7-fold and 2.0-fold compared to PE2 and PE3 systems, respectively, while improving edit/indel ratios by 3.4-fold in MMR-proficient cell types. Strategic installation of silent mutations near the intended edit can enhance prime editing outcomes by evading MMR. Prime editor protein optimization resulted in a PEmax architecture that enhances editing efficacy by 2.8-fold on average in HeLa cells. These findings enrich our understanding of prime editing and establish prime editing systems that show substantial improvement across 191 edits in seven mammalian cell types.

Sirtuin 1 Mediates Protection Against Delayed Cerebral Ischemia in Subarachnoid Hemorrhage in Response to Hypoxic Postconditioning.

Background Many therapies designed to prevent delayed cerebral ischemia (DCI) and improve neurological outcome in aneurysmal subarachnoid hemorrhage (SAH) have failed, likely because of targeting only one element of what has proven to be a multifactorial disease. We previously demonstrated that initiating hypoxic conditioning before SAH (hypoxic preconditioning) provides powerful protection against DCI. Here, we expanded upon these findings to determine whether hypoxic conditioning delivered at clinically relevant time points after SAH (hypoxic postconditioning) provides similarly robust DCI protection. Methods and Results In this study, we found that hypoxic postconditioning (8% O2 for 2 hours) initiated 3 hours after SAH provides strong protection against cerebral vasospasm, microvessel thrombi, and neurological deficits. By pharmacologic and genetic inhibition of SIRT1 (sirtuin 1) using EX527 and global Sirt1-/- mice, respectively, we demonstrated that this multifaceted DCI protection is SIRT1 mediated. Moreover, genetic overexpression of SIRT1 using Sirt1-Tg mice, mimicked the DCI protection afforded by hypoxic postconditioning. Finally, we found that post-SAH administration of resveratrol attenuated cerebral vasospasm, microvessel thrombi, and neurological deficits, and did so in a SIRT1-dependent fashion. Conclusions The present study indicates that hypoxic postconditioning provides powerful DCI protection when initiated at clinically relevant time points, and that pharmacologic augmentation of SIRT1 activity after SAH can mimic this beneficial effect. We conclude that conditioning-based therapies administered after SAH hold translational promise for patients with SAH and warrant further investigation.

Thrombolytic Enzymes of Microbial Origin: A Review.

Enzyme therapies are attracting significant attention as thrombolytic drugs during the current scenario owing to their great affinity, specificity, catalytic activity, and stability. Among various sources, the application of microbial-derived thrombolytic and fibrinolytic enzymes to prevent and treat vascular occlusion is promising due to their advantageous cost-benefit ratio and large-scale production. Thrombotic complications such as stroke, myocardial infarction, pulmonary embolism, deep venous thrombosis, and peripheral occlusive diseases resulting from blood vessel blockage are the major cause of poor prognosis and mortality. Given the ability of microbial thrombolytic enzymes to dissolve blood clots and prevent any adverse effects, their use as a potential thrombolytic therapy has attracted great interest. A better understanding of the hemostasis and fibrinolytic system may aid in improving the efficacy and safety of this treatment approach over classical thrombolytic agents. Here, we concisely discuss the physiological mechanism of thrombus formation, thrombo-, and fibrinolysis, thrombolytic and fibrinolytic agents isolated from bacteria, fungi, and algae along with their mode of action and the potential application of microbial enzymes in thrombosis therapy.

Restructuring the Gut Microbiota by Intermittent Fasting Lowers Blood Pressure.

[Figure: see text].

The gut microbiome contributes to blood-brain barrier disruption in spontaneously hypertensive stroke prone rats.

In recent years, it has become apparent that the gut microbiome can influence the functioning and pathological states of organs and systems throughout the body. In this study, we tested the hypothesis that the gut microbiome has a major role in the disruption of the blood-brain barrier (BBB) in the spontaneously hypertensive stroke prone rats (SHRSP), an animal model for hypertensive cerebral small vessel disease (CSVD). Loss of BBB is thought to be an early and initiating component to the full expression of CSVD in animal models and humans. To test this hypothesis, newly born SHRSP pups were placed with foster dams of the SHRSP strain or dams of the WKY strain, the control strain that does not demonstrate BBB dysfunction or develop hypertensive CSVD. Similarly, WKY pups were placed with foster dams of the same or opposite strain. The rationale for cross fostering is that the gut microbiomes are shaped by environmental bacteria of the foster dam and the nesting surroundings. Analysis of the bacterial genera in feces, using 16S rRNA analysis, demonstrated that the gut microbiome in the rat pups was influenced by the foster dam. SHRSP offspring fostered on WKY dams had systolic blood pressures (SBPs) that were significantly decreased by 26 mmHg (P < .001) from 16-20 weeks, compared to SHRSP offspring fostered on SHRSP dams. Similarly WKY offspring fostered on SHRSP dams had significantly increased SBP compared to WKY offspring fostered on WKY dams, although the magnitude of SBP change was not as robust. At ~20 weeks of age, rats fostered on SHRSP dams showed enhanced inflammation in distal ileum regardless of the strain of the offspring. Disruption of BBB integrity, an early marker of CSVD onset, was improved in SHRSPs that were fostered on WKY dams when compared to the SHRSP rats fostered on SHRSP dams. Although SHRSP is a genetic model for CSVD, environmental factors such as the gut microbiota of the foster dam have a major influence in the loss of BBB integrity.

SIRT1 mediates hypoxic preconditioning induced attenuation of neurovascular dysfunction following subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Vasospasm and delayed cerebral ischemia (DCI) contribute significantly to the morbidity/mortality associated with aneurysmal subarachnoid hemorrhage (SAH). While considerable research effort has focused on preventing or reversing vasospasm, SAH-induced brain injury occurs in response to a multitude of concomitantly acting pathophysiologic mechanisms. In this regard, the pleiotropic epigenetic responses to conditioning-based therapeutics may provide an ideal SAH therapeutic strategy. We previously documented the ability of hypoxic preconditioning (PC) to attenuate vasospasm and neurological deficits after SAH, in a manner that depends on the activity of endothelial nitric oxide synthase. The present study was undertaken to elucidate whether the NAD-dependent protein deacetylase sirtuin isoform SIRT1 is an upstream mediator of hypoxic PC-induced protection, and to assess the efficacy of the SIRT1-activating polyphenol Resveratrol as a pharmacologic preconditioning therapy. METHODS: Wild-type C57BL/6J mice were utilized in the study and subjected to normoxia or hypoxic PC. Surgical procedures included induction of SAH via endovascular perforation or sham surgery. Multiple endpoints were assessed including cerebral vasospasm, neurobehavioral deficits, SIRT1 expression via quantitative real-time PCR for mRNA, and western blot for protein quantification. Pharmacological agents utilized in the study include EX-527 (SIRT1 inhibitor), and Resveratrol (SIRT1 activator). RESULTS: Hypoxic PC leads to rapid and sustained increase in cerebral SIRT1 mRNA and protein expression. SIRT1 inhibition blocks the protective effects of hypoxic PC on vasospasm and neurological deficits. Resveratrol pretreatment dose-dependently abrogates vasospasm and attenuates neurological deficits following SAH - beneficial effects that were similarly blocked by pharmacologic inhibition of SIRT1. CONCLUSION: SIRT1 mediates hypoxic preconditioning-induced protection against neurovascular dysfunction after SAH. Resveratrol mimics this neurovascular protection, at least in part, via SIRT1. Activation of SIRT1 is a promising, novel, pleiotropic therapeutic strategy to combat DCI after SAH.

Prebiotics, Probiotics, and Acetate Supplementation Prevent Hypertension in a Model of Obstructive Sleep Apnea.

Disruption of the gut microbiota, termed gut dysbiosis, has been described in animal models of hypertension and hypertensive patients. We have shown that gut dysbiosis plays a causal role in the development of hypertension in a rat model of obstructive sleep apnea (OSA). Functional analysis of the dysbiotic microbiota in OSA demonstrates a loss of short chain fatty acid-producing bacteria. However, measurements of short chain fatty acid concentrations and testing of their role in blood pressure regulation are lacking. We hypothesized that reduced short chain fatty acids in the gut are responsible for OSA-induced hypertension. OSA significantly increased systolic blood pressure at 7 and 14 days ( P<0.05), an effect that was abolished by the probiotic Clostridium butyricum or the prebiotic Hylon VII. The 16S rRNA analysis identified several short chain fatty acid-producing bacteria that were significantly increased by Cbutyricum and Hylon treatment. Acetate concentration in the cecum was decreased by 48% after OSA ( P<0.05), an effect that was prevented by Cbutyricum and Hylon. Cbutyricum and Hylon reduced OSA-induced dysbiosis, epithelial goblet cell loss, mucus barrier thinning, and activation of brain microglia ( P<0.05 for each). To examine the role of acetate in OSA-induced hypertension, we chronically infused acetate into the cecum during 2 weeks of sham or OSA. Restoring cecal acetate concentration prevented OSA-induced gut inflammation and hypertension ( P<0.05). These studies identify acetate as a key player in OSA-induced hypertension. We demonstrate that various methods to increase cecal acetate concentrations are protective from the adverse effects of OSA on the microbiota, gut, brain, and blood pressure.

Detection of 224 candidate structured RNAs by comparative analysis of specific subsets of intergenic regions.

The discovery of structured non-coding RNAs (ncRNAs) in bacteria can reveal new facets of biology and biochemistry. Comparative genomics analyses executed by powerful computer algorithms have successfully been used to uncover many novel bacterial ncRNA classes in recent years. However, this general search strategy favors the discovery of more common ncRNA classes, whereas progressively rarer classes are correspondingly more difficult to identify. In the current study, we confront this problem by devising several methods to select subsets of intergenic regions that can concentrate these rare RNA classes, thereby increasing the probability that comparative sequence analysis approaches will reveal their existence. By implementing these methods, we discovered 224 novel ncRNA classes, which include ROOL RNA, an RNA class averaging 581 nt and present in multiple phyla, several highly conserved and widespread ncRNA classes with properties that suggest sophisticated biochemical functions and a multitude of putative cis-regulatory RNA classes involved in a variety of biological processes. We expect that further research on these newly found RNA classes will reveal additional aspects of novel biology, and allow for greater insights into the biochemistry performed by ncRNAs.

A novel fluorescent imaging technique for assessment of cerebral vasospasm after experimental subarachnoid hemorrhage.

Various techniques have been developed to study changes in the cerebral vasculature in numerous neuropathological processes including subarachnoid hemorrhage (SAH). One of the most widely employed techniques uses India ink-gelatin casting, which presents numerous challenges due to its high viscosity, rapid solidification, and its impact on immunohistochemical analysis. To overcome these limitations, we developed a novel technique for assessing cerebral vasospasm using cerebrovascular perfusion with ROX, SE (5-Carboxy-X-Rhodamine, Succinimidyl Ester), a fluorescent labeling dye. We found that ROX SE perfusion achieves excellent delineation of the cerebral vasculature, was qualitatively and quantitatively superior to India ink-gelatin casting for the assessment of cerebral vasospasm, permits outstanding immunohistochemical examination of non-vasospasm components of secondary brain injury, and is a more efficient and cost-effective experimental technique. ROX SE perfusion is therefore a novel and highly useful technique for studying cerebrovascular pathology following experimental SAH.

The lost language of the RNA World.

The possibility of an RNA World is based on the notion that life on Earth passed through a primitive phase without proteins, a time when all genomes and enzymes were composed of ribonucleic acids. Numerous apparent vestiges of this ancient RNA World remain today, including many nucleotide-derived coenzymes, self-processing ribozymes, metabolite-binding riboswitches, and even ribosomes. Many of the most common signaling molecules and second messengers used by modern organisms are also formed from RNA nucleotides or their precursors. For example, nucleotide derivatives such as cAMP, ppGpp, and ZTP, as well as the cyclic dinucleotides c-di-GMP and c-di-AMP, are intimately involved in signaling diverse physiological or metabolic changes in bacteria and other organisms. We describe the potential diversity of this "lost language" of the RNA World and speculate on whether additional components of this ancient communication machinery might remain hidden though still very much relevant to modern cells.

Bioinformatic analysis of riboswitch structures uncovers variant classes with altered ligand specificity.

Riboswitches are RNAs that form complex, folded structures that selectively bind small molecules or ions. As with certain groups of protein enzymes and receptors, some riboswitch classes have evolved to change their ligand specificity. We developed a procedure to systematically analyze known riboswitch classes to find additional variants that have altered their ligand specificity. This approach uses multiple-sequence alignments, atomic-resolution structural information, and riboswitch gene associations. Among the discoveries are unique variants of the guanine riboswitch class that most tightly bind the nucleoside 2'-deoxyguanosine. In addition, we identified variants of the glycine riboswitch class that no longer recognize this amino acid, additional members of a rare flavin mononucleotide (FMN) variant class, and also variants of c-di-GMP-I and -II riboswitches that might recognize different bacterial signaling molecules. These findings further reveal the diverse molecular sensing capabilities of RNA, which highlights the potential for discovering a large number of additional natural riboswitch classes.