An inducible germ cell ablation chicken model for high-grade germline chimeras.

Chicken embryos are a powerful and widely used animal model in developmental biology studies. Since the development of CRISPR technology, gene-edited chickens have been generated by transferring primordial germ cells (PGCs) into recipients after genetic modifications. However, low inheritance caused by competition between host germ cells and the transferred cells is a common complication and greatly reduces production efficiency. Here, we generated a gene-edited chicken, in which germ cells can be ablated in a drug-dependent manner, as recipients for gene-edited PGC transfer. We used the nitroreductase/metronidazole (NTR/Mtz) system for cell ablation, in which nitroreductase produces cytotoxic alkylating agents from administered metronidazole, causing cell apoptosis. The chicken Vasa homolog (CVH) gene locus was used to drive the expression of the nitroreductase gene in a germ cell-specific manner. In addition, a fluorescent protein gene, mCherry, was also placed in the CVH locus to visualize the PGCs. We named this system 'germ cell-specific autonomous removal induction' (gSAMURAI). gSAMURAI chickens will be an ideal recipient to produce offspring derived from transplanted exogenous germ cells.

Thalidomide and its metabolite 5-hydroxythalidomide induce teratogenicity via the cereblon neosubstrate PLZF.

Thalidomide causes teratogenic effects by inducing protein degradation via cereblon (CRBN)-containing ubiquitin ligase and modification of its substrate specificity. Human P450 cytochromes convert thalidomide into two monohydroxylated metabolites that are considered to contribute to thalidomide effects, through mechanisms that remain unclear. Here, we report that promyelocytic leukaemia zinc finger (PLZF)/ZBTB16 is a CRBN target protein whose degradation is involved in thalidomide- and 5-hydroxythalidomide-induced teratogenicity. Using a human transcription factor protein array produced in a wheat cell-free protein synthesis system, PLZF was identified as a thalidomide-dependent CRBN substrate. PLZF is degraded by the ubiquitin ligase CRL4CRBN in complex with thalidomide, its derivatives or 5-hydroxythalidomide in a manner dependent on the conserved first and third zinc finger domains of PLZF. Surprisingly, thalidomide and 5-hydroxythalidomide confer distinctly different substrate specificities to mouse and chicken CRBN, and both compounds cause teratogenic phenotypes in chicken embryos. Consistently, knockdown of Plzf induces short bone formation in chicken limbs. Most importantly, degradation of PLZF protein, but not of the known thalidomide-dependent CRBN substrate SALL4, was induced by thalidomide or 5-hydroxythalidomide treatment in chicken embryos. Furthermore, PLZF overexpression partially rescued the thalidomide-induced phenotypes. Our findings implicate PLZF as an important thalidomide-induced CRBN neosubstrate involved in thalidomide teratogenicity.

Brain structures and functional connectivity in neglected children with no other types of maltreatment.

Child maltreatment can adversely affect brain development, leading to vulnerabilities in brain structure and function and various psychiatric disorders. Among the various types of child maltreatment, neglect has the highest incidence rate (76.0%); however, data on its sole adverse influence on the brain remain limited. This case-control brain magnetic resonance imaging (MRI) study identified the changes in gray matter structure and function that distinguish neglected children with no other type of maltreatment (Neglect group, n = 23) from typically developing children (TD group, n = 140), and investigated the association between these structural and functional differences and specific psychosocial phenotypes observed in neglected children. Our results showed that the Neglect group had a larger right and left anterior cingulate cortex (R/L.ACC) and smaller left angular gyrus (L.AG) gray matter volume. The larger R/L.ACC was associated with hyperactivity and inattention. Resting-state functional analysis showed increased functional connectivity (FC) between the left supramarginal gyrus (L.SMG) in the salience network (SN) and the right middle frontal gyrus (R.MFG) simultaneously with a decrease in FC with the L.ACC for the same seed. The increased FC for the R.MFG was associated with difficulty in peer problems and depressive symptoms; a mediating effect was evident for depressive symptoms. These results suggest that the structural atypicality of the R/L.ACC indirectly contributes to the disturbed FCs within the SN, thereby exacerbating depressive symptoms in neglected children. In conclusion, exposure to neglect in childhood may lead to maladaptive brain development, particularly neural changes associated with depressive symptoms.

Language and sensory characteristics are reflected in voice-evoked responses in low birth weight children.

BACKGROUND: Children born with very low birth weight (VLBW) are at higher risk for cognitive impairment, including language deficits and sensorimotor difficulties. Voice-evoked response (P1m), which has been suggested as a language development biomarker in young children, remains unexplored for its efficacy in VLBW children. Furthermore, the relation between P1m and sensory difficulties in VLBW children remains unclear. METHODS: 40 children with VLBW were recruited at 5-to-6 years old (26 male, 14 female, mean age of months ± SD, 80.0 ± 4.9). We measured their voice-evoked brain response using child-customized magnetoencephalography (MEG) and examined the relation between P1m and language conceptual inference ability and sensory characteristics. RESULTS: The final sample comprised 36 children (23 boys, 13 girls; ages 61-86 months; gestational ages 24-36 weeks). As a result of multiple regression analysis, voice-evoked P1m in the left hemisphere was correlated significantly with language ability (ß = 0.414 P = 0.015) and sensory hypersensitivity (ß = 0.471 P = 0.005). CONCLUSION: Our findings indicate that the relation between P1m and language conceptual inference ability observed in term children in earlier studies is replicated in VLBW children, and suggests P1m intensity as a biomarker of sensory sensitivity characteristics. IMPACT: We investigated brain functions related to language development and sensory problems in very low birth-weight children. In very low birth weight children at early school age, brain responses to human voices are associated with language conceptual inference ability and sensory hypersensitivity. These findings promote a physiological understanding of both language development and sensory characteristics in very low birth weight children.

Dual Emission with Efficient Phosphorescence Promoted by Intermolecular Halogen Interactions in Luminescent Tetranuclear Zinc(II) Clusters.

The development of Zn-based phosphorescent materials, associated with a ligand-centered (LC) transition, is extremely limited. Herein, we demonstrated dual emissions including fluorescence and phosphorescence in luminescent tetranuclear Zn(II) clusters [Zn4LI4(µ3-OMe)2X2] (HLI = methyl-5-iode-3-methoxysalicylate; X = I, Br, Cl), incorporating iodine-substituted ligands. Single-crystal X-ray structural analyses and variable-temperature emission spectra studies revealed the presence of iodine substitutions, and intermolecular halogen interactions produced the internal/external heavy-atom effects and yielded strong green phosphorescence with a long emission lifetime (λmax = 510-522 nm, Φem = 0.28-0.47, τav = 0.78-0.95 ms, at 77 K). This work provided a new example that the introduction of halogen interactions is an advantageous approach for inducing phosphorescence in fluorescent metal complexes.

A ubiquitin-like protein encoded by the "noncoding" RNA TINCR promotes keratinocyte proliferation and wound healing.

Although long noncoding RNAs (lncRNAs) are transcripts that do not encode proteins by definition, some lncRNAs actually contain small open reading frames that are translated. TINCR (terminal differentiation-induced ncRNA) has been recognized as a lncRNA that contributes to keratinocyte differentiation. However, we here show that TINCR encodes a ubiquitin-like protein that is well conserved among species and whose expression was confirmed by the generation of mice harboring a FLAG epitope tag sequence in the endogenous open reading frame as well as by targeted proteomics. Forced expression of this protein promoted cell cycle progression in normal human epidermal keratinocytes, and mice lacking this protein manifested a delay in skin wound healing associated with attenuated cell cycle progression in keratinocytes. We termed this protein TINCR-encoded ubiquitin-like protein (TUBL), and our results reveal a role for TINCR in the regulation of keratinocyte proliferation and skin regeneration that is dependent on TUBL.

Reversible Transition between Discrete and 1D Infinite Architectures: A Temperature-Responsive Cu(I) Complex with a Flexible Disilane-Bridged Bis(pyridine) Ligand.

A thermoresponsive structural change based on a disilane-bridged bis(pyridine) ligand and CuI is reported. Single-crystal X-ray analysis revealed that there are two polymorphs in the Cu(I) complex: octanuclear copper(I) complex at 20 °C and 1D staircase copper(I) polymer complex at -173 °C. The formation of these polymorphs is due to the flexibility of the ligand. Cu-I bond formation is observed upon cooling the sample from -10 °C to -170 °C. The temperature-induced phase transition progression was clarified by DSC, VT-PXRD, and VT-photoluminescence measurements and indicated a reversible temperature-controlled crystal-to-crystal phase transition. Observation on a VT-stage using a high-speed camera showed crystal cracking during single-crystal to single-crystal transitions between these polymorphic forms.

Heterologous expression and characterization of salt-tolerant ß-glucosidase from xerophilic Aspergillus chevalieri for hydrolysis of marine biomass.

A salt-tolerant exo-ß-1,3-glucosidase (BGL_MK86) was cloned from the xerophilic mold Aspergillus chevalieri MK86 and heterologously expressed in A. oryzae. Phylogenetic analysis suggests that BGL_MK86 belongs to glycoside hydrolase family 5 (aryl-phospho-ß-D-glucosidase, BglC), and exhibits D-glucose tolerance. Recombinant BGL_MK86 (rBGL_MK86) exhibited 100-fold higher expression than native BGL_MK86. rBGL_MK86 was active over a wide range of NaCl concentrations [0%-18% (w/v)] and showed increased substrate affinity for p-nitrophenyl-ß-D-glucopyranoside (pNPBG) and turnover number (kcat) in the presence of NaCl. The enzyme was stable over a broad pH range (5.5-9.5). The optimum reaction pH and temperature for hydrolysis of pNPBG were 5.5 and 45 °C, respectively. rBGL_MK86 acted on the ß-1,3-linked glucose dimer laminaribiose, but not ß-1,4-linked or ß-1,6-linked glucose dimers (cellobiose or gentiobiose). It showed tenfold higher activity toward laminarin (a linear polymer of ß-1,3 glucan) from Laminaria digitata than laminarin (ß-1,3/ß-1,6 glucan) from Eisenia bicyclis, likely due to its inability to act on ß-1,6-linked glucose residues. The ß-glucosidase retained hydrolytic activity toward crude laminarin preparations from marine biomass in moderately high salt concentrations. These properties indicate wide potential applications of this enzyme in saccharification of salt-bearing marine biomass.

Examination of aminophenol-containing compounds designed as antiproliferative agents and potential atypical retinoids.

Retinoic acid (RA, 1), an oxidized form of vitamin A, binds to retinoic acid receptors (RAR) and retinoid X receptors (RXR) to regulate gene expression and has important functions such as cell proliferation and differentiation. Synthetic ligands regarding RAR and RXR have been devised for the treatment of various diseases, particularly promyelocytic leukemia, but their side effects have led to the development of new, less toxic therapeutic agents. Fenretinide (4-HPR, 2), an aminophenol derivative of RA, exhibits potent antiproliferative activity without binding to RAR/RXR, but its clinical trial was discontinued due to side effects of impaired dark adaptation. Assuming that the cyclohexene ring of 4-HPR is the cause of the side effects, methylaminophenol was discovered through structure-activity relationship research, and p-dodecylaminophenol (p-DDAP, 3), which has no side effects or toxicity and is effective against a wide range of cancers, was developed. Therefore, we thought that introducing the motif carboxylic acid found in retinoids, could potentially enhance the anti-proliferative effects. Introducing chain terminal carboxylic functionality into potent p-alkylaminophenols significantly attenuated antiproliferative potencies, while a similar structural modification of weakly potent p-acylaminophenols enhanced growth inhibitory potencies. However, conversion of the carboxylic acid moieties to their methyl esters completely abolished the cell growth inhibitory effects of both series. Insertion of a carboxylic acid moiety, which is important for binding to RA receptors, abolishes the action of p-alkylaminophenols, but enhances the action of p-acylaminophenols. This suggests that the amido functionality may be important for the growth inhibitory effects of the carboxylic acids.

Possible Role of Image-Enhanced Endoscopy in the Evaluation of Mucosal Healing of Ulcerative Colitis.

BACKGROUND: Mucosal healing (MH) is recognized as a therapeutic target in ulcerative colitis (UC) because of evidence that it is associated with favorable clinical outcomes. Current endoscopic assessment of MH by conventional white-light endoscopy is subject to several important clinical issues including the subjective nature of assessment, intra- and interobserver variability, and persistent microscopic inflammation, even in mucosa it was observed as quiescent on conventional endoscopy. SUMMARY: Advances in image-enhancement technologies enable the provision of high-contrast images that emphasize the mucosal structures, blood vessel patterns, and color tones of the intestinal mucosa, and recently, several image-enhanced endoscopy (IEE) techniques have become available for the assessment of MH in UC. Narrow-band imaging and dual-red imaging facilitate visualization of mucosal vascular structures, which is useful for detecting minor inflammation and predicting relapse because of the capturing of information on incomplete vascular regeneration in patients with UC. Linked-color imaging (LCI) is optimized to emphasize the redness of the mucosa and blood vessels, and is superior for depicting subtle color changes arising from mucosal inflammation. LCI could possibly be used to stratify UC patients with MH on conventional endoscopy. Autofluorescence imaging and i-scan can also depict subtle histological changes underlying the healing of mucosa in UC, revealing them as simple color changes. KEY MESSAGES: Accumulating evidence suggests that IEE techniques could overcome current unmet needs in the endoscopic assessment of MH in UC and contribute to improving therapy based on treat-to-target strategies.

Effects of Antibacterial Agents on Cancerous Cell Proliferation.

Myelosuppression, a side effect of anticancer drugs, makes people more susceptible to infectious diseases by compromising the immune system. When a cancer patient develops a contagious disease, treatment with an anticancer drug is suspended or postponed to treat the infectious disease. If there was a drug that suppresses the growth of cancer cells among antibacterial agents, it would be possible to treat both infectious diseases and cancer. Therefore, this study investigated the effect of antibacterial agents on cancer cell development. Vancomycin (VAN) had little effect on cell proliferation against the breast cancer cell, MCF-7, prostate cancer cell, PC-3, and gallbladder cancer cell, NOZ C-1. Alternatively, Teicoplanin (TEIC) and Daptomycin (DAP) promoted the growth of some cancer cells. In contrast, Linezolid (LZD) suppressed the proliferation of MCF-7, PC-3, and NOZ C-1 cells. Therefore, we found a drug that affects the growth of cancer cells among antibacterial agents. Next, when we examined the effects of the combined use of existing anticancer and antibacterial agents, we found VAN did not affect the growth suppression by anticancer agents. However, TEIC and DAP attenuated the growth suppression of anticancer agents. In contrast, LZD additively enhanced the growth suppression by Docetaxel in PC-3 cells. Furthermore, we showed that LZD inhibits cancer cell growth by mechanisms that involve phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway suppression. Therefore, LZD might simultaneously treat cancer and infectious diseases.

Partial Tracheal Defects Closure using Glutaraldehyde-Treated Autologous Pericardium.

BACKGROUND: The usefulness of autologous pericardium treated with glutaraldehyde (GA) for tracheal defect closure is unknown. This study preliminarily evaluated whether a GA-treated autologous pericardial graft can effectively close tracheal defects in a beagle model. METHODS: Defects of 10 mm × 10 mm were created on the trachea of 10 beagles and divided into a GA-treated group (n = 5), with tracheal reconstruction using GA-treated pericardium, and control group (n = 5), using fresh pericardium. Repair sites were evaluated through bronchoscopy and histology. Blood flows on graft were measured using laser Doppler technique on postoperative days (PODs) 0, 4, 7, 14, 28, and 56. Repair sites were histologically evaluated on POD 56. In addition, GA-treated pericardia of three other beagles were histologically evaluated 12 months postoperatively, for long-term follow-up. RESULTS: All animals survived; none developed anastomotic insufficiency. The mean suturing time and frequency of additional suture were significantly shorter and lower in the GA-treated group than in the control group (p = 0.002, 0.004). All animals in the control group exhibited graft contraction, whereas the GA-treated group healed with most graft residual and reepithelialization in the bronchoscopic and histological findings (p = 0.01, 0.004). Further, all long-term GA-treated pericardia of three beagles were confirmed as residual grafts with reepithelialization, without contraction, at 12 months postoperatively. Blood flows on graft using laser Doppler technique in the GA-treated group were detected at POD 14 or thereafter. CONCLUSION: GA-treated pericardium was easier to handle and provided favorable scaffolding, without graft contraction, compared with the nontreated pericardium at short- and long-term follow-up.

[Left Metastatic Lung Tumor for a Patient with Scimitar Syndrome:Report of a Case].

Scimitar syndrome is a subtype of partial anomalous pulmonary venous connection, a rare congenital disorder associated with hypoplasia of the right lung. In addition to the difficulty of isolated lung ventilation, resection of the left lung is associated with the risk of developing right heart failure due to increased right-to-left shunts. We report a case of a left lung metastasis of a patient with scimitar syndrome. The patient, a 58-year-old male, was diagnosed with scimitar syndrome at the age of 26 but had never experienced any symptoms. He underwent chemoradiotherapy for mid-pharynx carcinoma and achieved complete response. During follow-up, a nodule appeared in the lower lobe of the left lung. Since right heart catheterization revealed a pulmonary blood flow/systemic blood flow ratio (Qp/Qs) ratio of 2.6, intra-cardiac blood flow was diverted prior to pulmonary resection. Stanford type A acute aortic dissection occurred intra-operatively, and total aortic arch replacement was performed. Three months later, partial pulmonary resection was performed with extracorporeal membrane oxygenation (ECMO) on standby. As oxygenation was maintained by placing a blocker in the left lower lobe bronchus and ventilating the left upper lobe with high frequency jet ventilation, the operation was completed without using ECMO. The nodule was pathologically diagnosed as metastasis of mid-pharynx carcinoma. He did not develop heart failure and was discharged on post operated day 15.

[Ingenuity for the Uniportal Video-assisted Thoracic Surgery as a Minimally Invasive Procedure].

Uniportal video-assisted thoracic surgery (VATS) is a minimally invasive, wound-reducing approach performed mainly in Europe and Asia. This approach is rapidly gaining popularity in Japan. We performed a technique with layer awareness, grasping and dissection of tissue membrane even in uniport VATS as for open thoracotomy or multiport VATS. Interference is a problem with uniport VATS because surgical instruments are inserted and removed through a small incision of 4 cm or less;there-fore, instrument selection is critical. The use of curved forceps ensures more working space and reduced interference. The incision should be placed between the 4th or 5th intercostal space and should be 3.5 cm in size at our institution. For vascular manipulation, ligation and transection can be used when it is difficult to divide vessels with a stapler. During mediastinal lymph node dissection, a precise view can be achieved with the use of a custom-made spatula. Uniport VATS was performed in 51 cases from January 2019 to June 2022. Although recurrence was observed in two cases, no serious perioperative complications were observed, and the procedures were performed safely.

Prime editing in chicken fibroblasts and primordial germ cells.

CRISPR/Cas9-based genome editing technologies are revolutionizing developmental biology. One of the advanced CRISPR-based techniques is prime editing (PE), which enables precise gene modification in multiple model organisms. However, there has been no report of taking advantage of the PE system for gene editing in primordial germ cells (PGCs) thus far. In the current study, we describe a method to apply PE to the genome of chicken fibroblasts and PGCs. By combining PE with a transposon-mediated genomic integration, drug selection, and the single-cell culture method, we successfully generated prime-edited chicken fibroblasts and PGCs. The chicken PGC is widely used as an experimental model to study germ cell formation and as a vector for gene transfer to produce transgenic chickens. Such experimental models are useful in the developmental biology field and as potential bioreactors to produce pharmaceutical and nutritious proteins. Thus, the method presented here will provide not only a powerful tool to investigate gene function in germ cell development but also a basis for generating prime-edited transgenic birds.

Reversible and Stepwise Single-Crystal-to-Single-Crystal Transformation of a Platinum(II) Complex with Vapochromic Luminescence.

The vapochromic single-crystal-to-single-crystal (SCSC) transformation of a highly luminescent PtII complex bearing an N-heterocyclic carbene [Pt(CN)2 (tBu-impy)] (tBu-impyH+ =1-tert-butyl-3-(2-pyridyl)-1H-imidazolium) is reported. The trihydrate form of the complex, which exhibits blue 3 MMLCT emission owing to weak Pt⋅⋅⋅Pt interactions, changed its luminescence color from blue to yellowish-green upon the desorption of water molecules while keeping the high emission quantum yield of more than 0.45. Variable-temperature and continuous in-situ tracking of single-crystal X-ray diffraction measurements revealed that the SCSC transformation proceeds reversibly by the release and reabsorption of water molecules, thereby changing the stacked structure slightly. As a result, the dynamics of vapor-induced SCSC transformation were elucidated: that the anhydrous form returned to the original trihydrate form in a two-step process under a water vapor atmosphere. In addition, the PtII complex exhibited a similar SCSC response accompanied by a luminescence color change in the presence of methanol vapor, while being inactive toward ethanol vapor.

A Red-Light-Driven CO-Releasing Complex: Photoreactivities and Excited-State Dynamics of Highly Distorted Tricarbonyl Rhenium Phthalocyanines.

A complex comprising one [Re(CO)3 ]+ unit and a phthalocyanine (Pc) ligand (Re1 Pc) is shown to function as a photo-induced CO-releasing molecule (photoCORM) in the presence of O2 and a coordinative solvent under irradiation with red light, which can deeply penetrate living tissues. Transient absorption spectroscopic measurements indicate very short excited-state lifetimes and ultrafast intersystem crossing for Re1 Pc and Re2 Pc, which contains two [Re(CO)3 ]+ units. The excited-state properties are ascribed to efficient spin-orbit coupling and large Franck-Condon factors originating from the complexes' distorted structures, that is, unsymmetric coordination of [Re(CO)3 ]+ unit(s), one of which was confirmed by single-crystal X-ray analysis of a symmetrically substituted Pc with two [Re(CO)3 ]+ units. Re1 Pc represents a promising red-light-driven photoCORM that can be applied in biological environments or therapeutic applications.

A Series of D-A-D Structured Disilane-Bridged Triads: Structure and Stimuli-Responsive Luminescence Studies.

A series of σ-π extended octamethyltetrasilanes, which have phenothiazine, 9,9-dimethyl-9,10-dihydroacridine, or phenoxazine (1, 2, and 3) groups as donor moieties and thienopyrazine or benzothiadiazole (a and b) groups as acceptor fragments, has been prepared, and their optical properties have been studied as an extension of our work. All six compounds exhibited fluorescence in the solid state with maximum wavelengths centered in the range of 400 and 650 nm upon excitation by a UV lamp. Compound 2b showed apparent dual emission behavior in solution, which depends on solvent polarity, and a reversible photoluminescent change under mechanical and thermal stimuli in the solid state. Quantum chemical calculations suggest the contribution of a quasi-axial conformer of the 9,9-dimethyl-9,10-dihydroacridine moiety in 2b to the dual emission in solution and the mechanofluoroluminescence in the solid state, similarly to 1a. These studies provide new insight into the preparation of disilane-bridged triads capable of responding to multiple stimuli.

Envelopment by endothelial cells initiates translocation of avian primordial germ cell into vascular tissue.

BACKGROUND: In avian species, primordial germ cells (PGCs) migrate to the gonadal primordium through the vascular system. Because this mode of migration is reminiscent of cancer metastasis, it would be useful to elucidate the mechanisms underlying PGC migration via the bloodstream. Here, we sought to determine when, where, and how PGCs enter the vascular network by double visualization of PGCs and endothelial cells (ECs) in tie1:H2B-eYFP transgenic quails. RESULTS: In the left and right lateral germinal crescent regions corresponding to the anterior-most area vasculosa, more than 60% of PGCs were enveloped by differentiating ECs forming blood islands prior to vascular network formation. Cell morphology analysis suggested that the PGC-EC interaction was instructed by differentiating ECs. At a later developmental stage, ECs anastomosed to form a vascular network with a lumen that retained PGCs within it. As a consequence, many PGCs localized within the luminal space of the mature vascular network at later stages. CONCLUSIONS: Our findings demonstrate that the major type of avian PGC translocation into vascular tissue is not a typical intravasation, as performed by types of metastatic cancer cells, but rather a passive translocation (envelopment) mediated by differentiating ECs during early vasculogenesis.

[Surgical Treatment to Castleman's Disease after Embolization of Feeding Vessels].

A 48-year-old woman with an abnormal shadow on chest X-ray was referred to our institution. Contrast-enhanced chest computed tomography( CT) showed a large mass, 4.4 cm in diameter, in the right upper mediastinum. Castleman's disease was suspected, and several vessels flowing into the tumor were identified. Since severe intraoperative bleeding was expected, preoperative embolization of the feeding vessels was performed, followed by thoracotomy and tumor extirpation. The amount of blood loss was 50 ml. The pathological diagnosis was Castleman's disease, hyaline vascular type.