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Plasmodium vivax is the most widespread cause of malaria, especially in subtropical and temperate regions such as Asia-Pacific and America. P. vivax lactate dehydrogenase (PvLDH), an essential enzyme in the glycolytic pathway, is required for the development and reproduction of the parasite. Thus, LDH from these parasites has garnered attention as a diagnostic biomarker for malaria and as a potential molecular target for developing antimalarial drugs. In this study, we prepared a transformed Escherichia coli strain for the overexpression of PvLDH without codon optimization. We introduced this recombinant plasmid DNA prepared by insertion of the PvLDH gene in the pET-21a(+) expression vector, into the Rosetta(DE3), an E. coli strain suitable for eukaryotic protein expression. The time, temperature, and inducer concentration for PvLDH expression from this E. coli Rosetta(DE3), containing the original PvLDH gene, were optimized. We obtained PvLDH with a 31.0 mg/L yield and high purity (>95%) from this Rosetta(DE3) strain. The purified protein was characterized structurally and functionally. The PvLDH expressed and purified from transformed bacteria without codon optimization was successfully demonstrated to exhibit its potential tetramer structure and enzyme activity. These findings are expected to provide valuable insights for research on infectious diseases, metabolism, diagnostics, and therapeutics for malaria caused by P. vivax.

BACKGROUND: Due to the anatomical characteristics of the anterior epidural space, dorsal migration of material from herniated lumbar disc is quite rare. Also, bilateral foot drop due to unilateral dorsal migration of disc herniation is extremely rare. This report presents a case of sudden bilateral foot drop caused by the unilateral dorsal migration of material from a herniated lumbar disc. CASE DESCRIPTION: A 51-year-old male presented with sudden onset severe leg pain with bilateral foot drop. The patient was referred to our emergency department by a local clinic. Neurological examination showed bilateral ankle and big toe dorsiflexion weakness grade 1. There was no perianal anesthesia, anal sphincter weaknesses, or voiding difficulty. Apart from essential hypertension, this patient’s medical history was unremarkable. Magnetic resonance imaging showed that intervertebral disc material in the dorsal extradural space at the L3-4 level had compressed the dural sac from the left side to the center. We performed an emergency operation. The pathologic result revealed fibrous cartilaginous materials. After the operation, the leg pain was markedly resolved. At postoperative three months, the patient showed improvement of foot drop. CONCLUSION: We recommended emergent mass removal, which produced a favorable outcome.

This study investigated the occurrence and distribution of microplastics utilizing zooplankton samples collected in the Western Indian Ocean because there is no information concerning epipelagic zone in the open ocean. We collected microplastics from three water layers [surface mixed layer(SML), middle layer(ML), lower layer(LL)] within 200 m using a Multiple Opening/Closing Net and Environmental Sensing (opening: 1 ㎡) at 22 stations of 1 degree interval between 5°N and 16°S along the 67°E of Western Indian Ocean in 2017. The microplastics were consistently found in almost all samples and the microplastic abundance ranged between 0.00-2.01 particles/㎥ from the 3 layers. And the average microplastic abundance was highest in the lower layer (0.30±0.09 particles/㎥) and lowest in middle layer (0.26±0.08 particles/㎥). The percentage of fiber was highest in the SML (55.7%) and the LL (45.9%), and the percentage of film was highest in ML (46.8%). The microplastic abundance in the size of 1.0-5.0 ㎜ was highest in SML (42.0%), while the abundance in the size of 0.2-0.5 ㎜ was highest in ML(56.8%) and LL(54.5%). The stations can be divided into four sections including upwelling characterized by Seychelles-Chagos Thermal Ridge (SCTR) based on the 20℃-isotherm depth (D20). The average microplastic abundance was the highest in SML (0.23±0.06 particles/㎥) in 1°S~5°S, and in LL (0.50±0.25 particles/㎥) at latitudes of 10°S~16°S and in LL (0.32±0.16 particles/㎥) at latitudes between 5°N~EQ. However, the average microplastic abundance at latitudes of 6°S ~9°S corresponding to the upwelling zone was highest in the ML (0.65±0.38 particles/㎥) with the high percentage of film (68.7%). Cluster analysis by microplastics occurred in each water layers showed that the stations were divided into 3 groups in each layer. Groups in SML and LL were mainly clustered by fiber, whereas groups in ML was mainly clustered by film, which was associated with the upwelled region of Seychelles-Chagos Thermal Ridge (SCTR). Fourier transform infrared spectroscopy analysis showed that the main types of microplastics were dominated by fiber (40.6%) and film (73.2%) characterized by polycarbonate. Present results showed that meridional and vertical distribution of microplastics in the epipelagic zone varied with the physical characteristics of upwelling zone characterized by Seychelles-Chagos Thermal Ridge (SCTR) in the Western Indian Ocean.

Anterior approach cervical surgery is widely used for accessing C3 lesions. When operating with an anterior approach, the surgical field is obstructed by mandible. Neck extension is popular method to secure better surgical field but risk devastating neurological damage. To overcome this limited surgical field without neck extension, we adopted nasotracheal intubation and evaluated its efficiency.We retrospectively analyzed 16 patients who underwent anterior cervical discectomy or corpectomy of C3 lesions via nasotracheal intubation. We enrolled an additional 29 patients who underwent anterior cervical discectomy or corpectomy of C3 lesions via orotracheal intubation as a control group. All patients had been diagnosed with cervical spondylotic myelopathy or ossification of the posterior longitudinal ligament. We measured the mandibular-cervical angle, which is the angle between the lower mandibular line and anterior vertebral line.The mandibular-cervical angle was increased by 7.3 with nasotracheal intubation compared to orotracheal intubation.Nasotracheal intubation is an effective surgical option for securing the surgical field without neck extension in anterior cervical surgery including C3 lesions.

Bandgap engineering of compound semiconductors and the fabrication of bandgap-modulated heterostructures are important for enabling the development of modern optoelectronics. However, these engineering processes are challenging for two-dimensional (2D) semiconductors of transition metal dichalcogenides, particularly on a large scale. Herein, we report the wafer-scale homogeneous growth of composition-modulated WS2xSe2(1-x) alloys with a continuously tunable bandgap using metal–organic chemical vapor deposition. Well-optimized growth produces monolayer films with excellent homogeneity over the entire wafer. The substitutional atomic chalcogen (S, Se) concentration in WS2xSe2(1-x) alloys is precisely controlled by varying the flow rate of the metal–organic precursors, leading to a bandgap modulation from 1.67 to 2.05 eV, as determined from absorbance spectra. Notably, the optical bandgap of WS2xSe2(1-x) alloys exhibits a nearly linear relationship with the chalcogen composition, implying a low bowing effect. This bowing-alleviated bandgap modulation is attributed to the small lattice mismatch, strain relaxation, and thermodynamic miscibility in the WS2xSe2(1-x) alloys, as confirmed by density-functional theory calculations. Furthermore, the fabrication of hetero-multilayers by stacking differently alloyed films is demonstrated. The produced heterostructure film exhibits a broad spectral absorbance distinct from that of the individual layers. The findings of this study provide insights for the advancement of versatile design of functional 2D optoelectronics.

Biodiversity has been declining and extinction rates have been exponentially increasing because of land use changes, invasion of exotic species, nutrient enrichment and climate change. In this scenario, many international networks such as the International Union for the Conservation of Nature have been making efforts to raise conservation awareness and preserve species and their habitats in many countries. The relict longhorn beetleCallipogon relictusSemenov, 1899 (Coleoptera: Cerambycidae) is the largest coleopteran species in the Palearctic region and has a unique distribution compared to its congeneric species.Callipogon relictushas been protected by two Korean laws since it was designated as a Korean Natural Monument and an Endangered Species in 1968 and 2012, respectively. To improve the conservation of this species, ecological and biological data were obtained from studies performed during the last 12 years on its populations in Gwangneung Forest, the fourth UNESCO biosphere reserve in South Korea. Previously scattered distribution records ofC. relictusfrom South Korea from 1932 to 2007 are therefore summarized and ecological features of adults observed during fields studies performed from 2008 to 2019 are presented. Based on the summarized data, we suggest different management measures and conservation efforts to maintain the size ofC. relictuspopulations in South Korea, which can also be further used in the restoration of other endangered insects.

BACKGROUND Identifying genetic mutations in cancer patients have been increasingly important because distinctive mutational patterns can be very informative to determine the optimal therapeutic strategy. Recent studies have shown that deep learning-based molecular cancer subtyping can be performed directly from the standard hematoxylin and eosin (H&E) sections in diverse tumors including colorectal cancers (CRCs). Since H&E-stained tissue slides are ubiquitously available, mutation prediction with the pathology images from cancers can be a time- and cost-effective complementary method for personalized treatment. AIM To predict the frequently occurring actionable mutations from the H&E-stained CRC whole-slide images (WSIs) with deep learning-based classifiers. METHODS A total of 629 CRC patients from The Cancer Genome Atlas (TCGA-COAD and TCGA-READ) and 142 CRC patients from Seoul St. Mary Hospital (SMH) were included. Based on the mutation frequency in TCGA and SMH datasets, we chose APC, KRAS, PIK3CA, SMAD4, and TP53 genes for the study. The classifiers were trained with 360 × 360 pixel patches of tissue images. The receiver operating characteristic (ROC) curves and area under the curves (AUCs) for all the classifiers were presented. RESULTS The AUCs for ROC curves ranged from 0.693 to 0.809 for the TCGA frozen WSIs and from 0.645 to 0.783 for the TCGA formalin-fixed paraffin-embedded WSIs. The prediction performance can be enhanced with the expansion of datasets. When the classifiers were trained with both TCGA and SMH data, the prediction performance was improved. CONCLUSION APC, KRAS, PIK3CA, SMAD4, and TP53 mutations can be predicted from H&E pathology images using deep learning-based classifiers, demonstrating the potential for deep learning-based mutation prediction in the CRC tissue slides.

The influences of physiochemical processes on the distribution of surface chlorophyll-a (Chl-a) and subsurface chlorophyll maximum (SCM) were investigated in the southeastern Yellow Sea (YS) during summer in 2010–2013. The paths of typhoons from 2010 to 2012 were different, resulting in different patterns of surface Chl-a and the SCM layer. Surface Chl-a appeared in high concentrations at the coastal area in 2010 and 2012, and at all stations in 2011. The SCM layer descended in the central area in 2012 and rose in the coastal area in 2010, and lifted at all stations in 2011. The SCM layer was shallower from the open sea located at the Yellow Sea Cold Water to the coastal area due to tidal fronts and topographic properties. As a result, the distribution characteristics of Chl-a concentration according to typhoon paths and water masses may affect the marine ecosystem in the YS.

The retinoic-acid inducible gene (RIG)-I is a cytoplasmic pattern recognition receptor that senses single-stranded (ss) or double-stranded (ds) RNA. RIG-I also senses AT-rich dsDNA, poly(dA:dT), through the action of an RNA polymerase III-transcribed RNA intermediate. Upon the binding of an RNA ligand, RIG-I binds to the mitochondrial antiviral-signaling protein (MAVS) and induces the formation of filamentous aggregates of MAVS, leading to the formation of a signaling complex that drives Type I interferon (IFN) responses. In the current study, we investigated the issue of whether the SUMOylation of MAVS induced by poly(dA:dT) affects the aggregation of MAVS in the RIG-I/MAVS pathway in human keratinocytes. Our results show that the poly(dA:dT)-induced secretion of IFN-β was dependent on RIG-I and MAVS. The inhibition of SUMOylation by Ginkgolic acid or Ubc9 siRNA was found to inhibit the poly(dA:dT)-induced secretion of IFN-β, suggesting that the SUMOylation is required for the poly(dA:dT)-activated RIG-I/MAVS pathway, which drives the secretion of IFN-β. In addition, treatment with poly(dA:dT) enhanced the formation of polymeric chains of small-ubiquitin like modifiers (SUMO)3, but not SUMO1 and SUMO2, on MAVS. Our results also show that the conjugation of SUMO3 to MAVS induced by poly (dA:dT) enhanced the aggregation of MAVS. These collective results show that the formation of SUMO3-conjugated chains of MAVS induced by poly (dA:dT), a ligand of RIG-I, enhances the aggregation of MAVS which, in turn, drives the secretion of IFN-β in human keratinocytes.

We report analysis of phosphatase activity and inhibition on droplet-based microfluidic chips. Phosphatases are such attractive potential drug targets because abnormal phosphatase activity has been implicated in a variety of diseases including cancer, neurological disorders, diabetes, osteoporosis, and obesity. So far, several methods for assessing phosphatase activity have been reported. However, they require a large sample volume and additional chemical modifications such as fluorescent dye conjugation and nanomaterial conjugation, and are not cost-effective. In this study, we used an artificial phosphatase substrate 3-O-methylfluorescein phosphate as a fluorescent reporter and dual specificity phosphatase 22. Using these materials, the phosphatase assay was performed from approximately 340.4 picoliter (pL) droplets generated at a frequency of ~40 hertz (Hz) in a droplet-based microfluidic chip. To evaluate the suitability of droplet-based platform for screening phosphatase inhibitors, a dose–response inhibition study was performed with ethyl-3,4-dephostatin and the half-maximal inhibitory concentration (IC50) was calculated as 5.79 ± 1.09 μM. The droplet-based results were compared to microplate-based experiments, which showed agreement. The droplet-based phosphatase assay proposed here is simple, reproducible, and generates enormous data sets within the limited sample and reagent volumes.

Interleukin (IL)-18, a member of the IL-1 family, has been implicated in the development of a variety of inflammatory skin diseases. The binding of double-stranded (ds) DNA to absent in melanoma (AIM) 2 induces the oligomerization of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and the recruitment of pro-caspase-1, which leads to the activation of caspase-1, subsequently resulting in the secretion of IL-1β and IL-18. Curcumin is a naturally occurring polyphenolic compound in Curcuma longa and is also well known to have anti-oxidant and anti-inflammatory properties. In the current study, we focused on the inhibitory effect of curcumin on ASC oligo-merization in human keratinocytes. The IL-18 level was measured by using an ELISA kit. The protein levels of AIM2 inflammasome components, such as AIM2, ASC, and pro-caspase-1, were determined by using a western blot. The level of ASC oligomerization was also determined by using a western blot. Our results show pre-treatment with curcumin attenuated poly (dA:dT)-induced secretion of IL-18 in IFN-γ-primed human keratinocytes. Pre-treatment with 10 µM curcumin was also found to inhibit ASC oligomerization in IFN-γ-primed cells. These collective results show that curcumin inhibits the poly (dA:dT)-induced secretion of IL-18 via the suppression of ASC oligomerization in IFN-γ-primed human keratinocytes.

We present the case of a 38-year-old male who complained of repeated dizziness and syncope. Rotational vertebral artery syndrome (RVAS) was diagnosed via videonystagmoraphy (VNG), computed tomography angiography (CTA) and three-position digital subtraction angiography (DSA). In the neutral position, CTA and DSA revealed left vertebral artery (VA) stenosis at the C2 transverse foramen and right VA hypoplasia. When the head was turned to the right, the blood flow stopped at the C2 level. The bony structure around the VA at the C2 transverse foramen was decompressed via an anterior surgical approach, and the symptoms resolved. This case present the precise stenotic point evaluation by three-position DSA is crucial for the planning of surgical treatment.

The use of alternative biocides has increased due to their economic and ecological relevance. Although data regarding the toxicity of commercial alternative biocides in marine organisms are accumulating, little is known about their toxic pathways or mechanisms. To compare the toxic effects of commercial alternative biocides on non-target pelagic fish (flounder) embryos, we investigated the adverse effects of developmental malformation and transcriptional changes. Three biocides including Diuron, Irgarol 1051® and Sea-Nine 211® produced a largely overlapping suite of developmental malformations, including tail-fin fold defects and dorsal body axis curvature. In our test, the potencies of these biocides were ranked in the following order with respect to malformation and mortalities: Sea-Nine 211® > Irgarol 1051® > Diuron. Consistent with the toxicity rankings, the expression of genes related to heart formation was greater in embryonic flounder exposed to Sea-Nine 211® than in those exposed to Irgarol 1051® or Diuron, while expression of genes related to fin malformation was greater in the Irgarol 1051® exposure group. In analyses of differential gene expression (DEG) profiles (fold change of genes with a cutoff P

We investigated the distributional pattern of mesozooplankton and the related environmental characteristics across the frontal zone in the South Sea of Korea during spring and autumn 2016. The frontal zone (FZ) was formed by Korean Southern Coastal Waters and Tsushima Warm Current, and the physical structure of front was more distinct during autumn than spring. The peaks of mesozooplankton abundance were not coincident (p > 0.05) with peaks of integrated chlorophyll-a (chl-a) concentrations (spring: chl-a = 44.5 mg m−2 at FZ, mesozooplankton = 8,198 inds.m−3 at coastal zone (CZ); autumn: chl-a=141.2 mg m−2 at CZ, mesozooplankton = 5,412 inds.m−3 at FZ). Cluster analysis divided the mesozooplankton community into FZ, CZ and offshore zone (OZ) mainly by the dominant copepods (adult and immature) during both seasons that was associated with chl-a based on principal component analysis. All the zones were characterized by temporal difference in occurrence of copepods, which can be explained by the cyclopoid-to-calanoid (Cy:Ca) ratio. Cy:Ca ratio was dependent mainly on the co-occurrence of higher concentrations of chl-a and cyclopoids at each zone between spring and autumn. Paracalanus copepodites dominated at all stations across the FZ during both periods. Cy:Ca ratio at FZ in spring showed a distinctively higher value than that of CZ and OZ, whereas the pattern was the opposite in autumn. These results indicated that the temporal distribution of mesozooplankton across the frontal zone in the South Sea was mainly characterized by the zonal characteristics of FZ, unlike CZ and OZ, and was the result of temporal differences in chl-a concentrations and the associated increase of cyclopoid copepods (Oithona spp.).

Long-term population monitoring is very important for the conservation of threatened and endangered species. The Black-faced Spoonbill Platalea minor is a globally endangered species and the majority of its population breed on uninhabited islands off the west coast of the Korean Peninsula. The Chilsan Archipelago in the southwest is the southernmost breeding site for this species in Korea and is expected to help buffer the potential dispersal of Black-faced Spoonbills from breeding populations currently concentrated in the Gyeonggi Bay area. As there has been a lack of information regarding the number of Black-faced Spoonbills, their characteristics, and their recent population trends, the present study investigated the population trends of Black-faced Spoonbills breeding in the Chilsan Archipelago. The study, from 2014 to 2018, examined breeding success in 2015 and 2018, and the size and location of nests in 2018. The number of nests ranged from 43 to 163 (2014, 43; 2015, 93; 2016, 84; 2017, 85; 2018, 163). The percentage of birds that attempted breeding between April and May was 84.9% in 2015 and 87.1% in 2018, while the breeding success rate was 69.9% in 2015 and 81.5% in 2018. The average nest size in 2018 was 55.06±6.09 cm long and 53.44±6.56 cm wide, while the average surface slope below the nest was 25.42±6.95°. In total, nine plant species were used as vegetation for nests. During the present study, the environmental conditions and the presence of anthropogenic disturbances seemed to affect breeding success. Therefore, strictly controlled access to breeding sites and effective management are needed to protect and conserve breeding populations of this endangered species.

The Seychelles–Chagos Thermocline Ridge (SCTR), is formed by strong air-ocean interactions that cause open-ocean upwelling in the western Indian Ocean (WIO). The SCTR enhances upper-ocean nutrient levels, which promotes phytoplankton growth, leading to high zooplankton abundance and a strong nursery habitat for fish. To evaluate the evidence of significant upwelling in the SCTR, upwelling indicators associated with variability in the mesozooplankton community and other environmental factors were compared between SCTR and non-SCTR regions. We investigated mesozooplankton and physicochemical factors using multiple opening/closing net and environmental sensing system at three epipelagic (0–200 m) depth strata along 67°E (16°S–5°N) in the central and WIO. Survey stations were clustered into six environmental regions, including SCTR regions (I and II) and non-SCTR regions of the Southwest Monsoon Currents (SMC), Cold-Core Eddy (CCE), and the South Equatorial Current regions North(N) and South(S). Higher abundances of mesozooplankton were observed in the surface mixed layer (SML) (mean: 1275 inds. m−3). In the SML, Noctiluca scintillans were distributed ubiquitously, regions in the CCE were significantly different (p

An investigation on the habitation of Callipogonrelictus Semenov, 1899 in Gwangneung forest was carried out, where the Korea National Arboretum is located. In an investigation spanning the last eleven years (2007–2017), 22 emergence holes, nine pupal chambers, six adults, and two larvae of C.relictus were identified. In this study, biological information about habitation of C.relictus is provided by comparing and combining the results of this investigation with a survey conducted in Ussurisky Nature Reserve, Russia, in 2015. The distribution is also reviewed to include the Korean Peninsula and a new location of South Korea is added to the distribution for C.relictus.

Since the ban of tributyltin in antifouling paints, many alternative biocides have been introduced to prevent settlement and growth of marine organisms on ship hulls. Zinc pyrithione (ZnPT) is one of the most frequently used alternative biocides in antifouling paints. This paper reviewed the overall chemical properties, toxicological characteristics, and environmental fates of ZnPT, as well as the analytical challenges of studying pertinent processes. ZnPT is generally toxic to a wide range of marine organisms, including algae, bivalves, sea urchins, polychaetes, crustaceans, and fish, typically at μg/L levels. ZnPT can be transchelated into other compounds in the presence of metal ions, and photodegrades when exposed to UV light. ZnPT is also reported to be biodegraded or hydrolyzed forming several metabolites of their own toxicity and stability. However, ZnPT accumulates in the water column or sediment, if it does not degrade at certain environmental conditions. To determine potential risks caused by ZnPT in the marine environment, studies have evaluated the environmental distribution of ZnPT with various chromatographic or voltammetry methods. Unfortunately, rapid transchelation and degradation of ZnPT in both the marine environment and laboratory interfered with most of the methods employed, making it difficult to evaluate its environmental distribution. More robust and sensitive analytical methods need to be developed to reliably describe the environmental release and distribution of ZnPT. To comprehensively understand the risk posed by the input of ZnPT into the marine environment, total degradation processes and its potential products also need to be adequately addressed.

The longhorn beetle genus Callipogon Audinet-Serville represents a small group of large wood-boring beetles whose distribution pattern exhibits a unique trans-Pacific disjunction between the East Asian temperate rainforest and the tropical rainforest of the Neotropics. To understand the biogeographic history underlying this circum-Pacific disjunct distribution, we reconstructed a molecular phylogeny of the subfamily Prioninae with extensive sampling of Callipogon using multilocus sequence data of 99 prionine and four parandrine samples (ingroups), together with two distant outgroup species. Our sampling of Callipogon includes 18 of the 24 currently accepted species, with complete representation of all species in our focal subgenera. Our phylogenetic analyses confirmed the purported affinity between the Palearctic Callipogon relictus and its Neotropical congeners. Furthermore, based on molecular dating under the fossilized birth-death (FBD) model with comprehensive fossil records and probabilistic ancestral range reconstructions, we estimated the crown group Callipogon to have originated in the Paleocene circa 60 million years ago (Ma) across the Neotropics and Eastern Palearctics. The divergence between the Palearctic C. relictus and its Neotropical congeners is explained as the result of a vicariance event following the demise of boreotropical forest across Beringia at the Eocene-Oligocene boundary. As C. relictus represents the unique relictual species that evidentiates the lineage's expansive ancient distribution, we evaluated its conservation importance through species distribution modelling. Though we estimated a range expansion for C. relictus by 2050, we emphasize a careful implementation of conservation programs towards the protection of primary forest across its current habitats, as the species remains highly vulnerable to habitat disturbance.

Euphausia pacifica (E. pacifica) is an important food source for fish and other organisms in marine ecosystems. We analyzed the carbon- and nitrogen-stable isotope ratios in potential diet sources to determine the feeding characteristics of E. pacifica in the Yellow Sea Cold Water Mass (YSCWM), a habitat for oversummering. E. pacifica was collected in the spring and summer from the central and coastal parts of the Yellow Sea from 2012 to 2014, and potential prey were collected in summer 2014. Although the δ13C values of E. pacifica differed significantly in spring each year, they narrowed to a specific range in summer. The δ15N values were always 2‰ higher in summer than in spring, suggesting that the diet of E. pacifica inhabiting the YSCWM was limited. In coastal stations, the contribution of any major food source calculated using an isotope mixing model was not more than 20%, suggesting that E. pacifica is omnivorous. However, at two stations in the YSCWM, 2.0–5.0-mm and > 5-mm plankton contributed 42 and 22% of their diet on average, respectively. These results indicate that E. pacifica inhabiting waters affected by cold water feeds on relatively large zooplankton such as the copepods Calanus sinicus. Therefore, the YSCWM likely plays a role in the trophic shift of E. pacifica during the summer.