Cysts of the ligamentum flavum are often linked to ischemic conditions: A morphological study.

"Cysts of the ligamentum flavum (cysts-LF)" is the term for non-neoplastic cystic lesion involving LF. The aim of the present study was to elucidate the histopathological characteristics and pathogenesis of "cysts-LF". Herein, we defined cysts-LF as spinal cysts containing degenerative LF components. From archival cases, we investigated 18 symptomatic cysts-LF surgically removed from 18 patients (13 males and five females; median age 68.5 years [range, 42-86 years]). The elastic fibers of LF components in the wall were separated and/or torn, and cyst walls were accompanied by chondroid metaplasia (17 cases), myxoid changes (13 cases), ossification (11 cases), amyloid deposits (14 cases), hemosiderosis (six cases), granular/smudgy calcification (four cases), synovial cell linings (three cases), and severe inflammatory infiltrates (one case). These histologic features of our cysts-LF were shared by previously reported "cysts-LF." Fourteen cysts-LF demonstrated vascular stenosis/occlusion, and eight showed thick hyalinized vessels, suggesting local circulatory insufficiency. Eight cases (44%) exhibited lipomembranous fat necrosis, accompanied by hyalinized vascular changes (p = 0.003). Ischemic conditions were observed in nearly half of the present cysts-LF, and may be one of the main contributing factors for the formation of cysts-LF, via degeneration and cystic changes in the LF.

Improved Electrochemical Peptide Synthesis Enabled by Electron-Rich Triaryl Phosphines.

While remarkable progress has been made in the development of peptide medicines, many problems related to peptide synthesis remain unresolved. Previously, we reported electrochemical peptide synthesis using a phosphine as a potentially recyclable coupling reagent. However, there was room for improvement from the point of view of reaction efficiency, especially in the carboxylic acid activation step and the peptide bond formation step. To overcome these challenges, we searched for the optimal phosphine. Among phosphines with various electronic properties, we found that electron-rich triaryl phosphines improved the reaction efficiency. Consequently, we successfully performed electrochemical peptide synthesis on sterically hindered and valuable amino acids. We also synthesized oligopeptides that were challenging with our previous method. Finally, we examined the effect of substituents on the phosphine cations, and gained some insights into reactivity, which will aid researchers designing reactions involving phosphine cations.

Suppression of TNF-α activity by immobilization rescues Mkx expression and attenuates tendon ossification in a mouse Achilles tenotomy model.

Traumatic heterotopic ossification is a condition in which extraskeletal bone formation occurs in soft tissues after injury. It most commonly occurs in patients who had major orthopedic surgery and in those with severe extremity injuries. The lesion causes local pain and can impair motor function of the affected limb, but there is currently no established prophylaxis or treatment for this condition. In this study, we show that immobilization at an early stage of the inflammatory response after injury can attenuate ossification formation in a murine Achilles tenotomy model. Gene expression analysis revealed a decrease in the expression of Tnf and an increase in the expression of Mkx, which encodes one of the master regulators of tendon differentiation, Mohawk. Notably, we found that TNF-α suppressed the expression of Mkx transcripts and accelerated the osteogenic differentiation of tendon-derived mesenchymal stem cells (MSCs), suggesting that TNF-α acts as a negative regulator of Mkx transcription. Consistent with these findings, pharmaceutical inhibition of TNF-α increased the expression of Mkx transcripts and suppressed bone formation in this mouse model. These findings reveal the previously unrecognized involvement of TNF-α in regulating tendon MSC fate through suppression of Mkx expression and suggest that TNF-α is a potential target for preventing traumatic heterotopic ossification.

Evidence of causality of low body mass index on risk of adolescent idiopathic scoliosis: a Mendelian randomization study.

Introduction: Adolescent idiopathic scoliosis (AIS) is a disorder with a three-dimensional spinal deformity and is a common disease affecting 1-5% of adolescents. AIS is also known as a complex disease involved in environmental and genetic factors. A relation between AIS and body mass index (BMI) has been epidemiologically and genetically suggested. However, the causal relationship between AIS and BMI remains to be elucidated. Material and methods: Mendelian randomization (MR) analysis was performed using summary statistics from genome-wide association studies (GWASs) of AIS (Japanese cohort, 5,327 cases, 73,884 controls; US cohort: 1,468 cases, 20,158 controls) and BMI (Biobank Japan: 173430 individual; meta-analysis of genetic investigation of anthropometric traits and UK Biobank: 806334 individuals; European Children cohort: 39620 individuals; Population Architecture using Genomics and Epidemiology: 49335 individuals). In MR analyses evaluating the effect of BMI on AIS, the association between BMI and AIS summary statistics was evaluated using the inverse-variance weighted (IVW) method, weighted median method, and Egger regression (MR-Egger) methods in Japanese. Results: Significant causality of genetically decreased BMI on risk of AIS was estimated: IVW method (Estimate (beta) [SE] = -0.56 [0.16], p = 1.8 × 10-3), weighted median method (beta = -0.56 [0.18], p = 8.5 × 10-3) and MR-Egger method (beta = -1.50 [0.43], p = 4.7 × 10-3), respectively. Consistent results were also observed when using the US AIS summary statistic in three MR methods; however, no significant causality was observed when evaluating the effect of AIS on BMI. Conclusions: Our Mendelian randomization analysis using large studies of AIS and GWAS for BMI summary statistics revealed that genetic variants contributing to low BMI have a causal effect on the onset of AIS. This result was consistent with those of epidemiological studies and would contribute to the early detection of AIS.

Eribulin mesylate induces bone mass loss by promoting osteoclastic bone resorption in mice.

Over the past few decades, the clinical outcomes of patients with cancer have significantly improved mostly owing to the development of effective chemotherapeutic treatments. However, chronic health conditions such as bone mass loss and risk of fragility fractures caused by chemotherapy have also emerged as crucial issues in patients treated for cancer. In this study, we aimed to understand the effect of eribulin mesylate (ERI), a microtubule-targeting agent currently used to treat metastatic breast cancer and certain subtypes of advanced sarcomas, on bone metabolism in mice. The administration of ERI reduced bone mass in mice, mainly by promoting osteoclast activity. Gene expression analysis of skeletal tissues revealed no change in the expression levels of the transcripts for RANK ligand, one of the master regulators of osteoclastogenesis; however, the transcript levels of osteoprotegerin, which neutralizes RANK ligand, were significantly reduced in ERI-treated mice compared with those in vehicle-treated controls, indicating a relative increase in RANK ligand availability after ERI treatment. In line with the increased bone resorption in ERI-treated mice, we found that zoledronate administration effectively suppressed bone loss in these mice. These results reveal a previously unrecognized effect of ERI on bone metabolism and suggest the application of bisphosphonates for patients with cancer undergoing treatment with ERI.

Condoliase chemonucleolysis for lumbar disc herniation: A post-hoc follow-up study of patients in previous clinical trials.

BACKGROUND: Chemonucleolysis with condoliase significantly improved clinical symptoms in patients with lumbar disc herniation. We evaluated the surgical intervention rate and outcomes for >1 year after condoliase treatment. METHODS: This was a follow-up study of patients who received condoliase or placebo in two previous randomized, placebo-controlled clinical trials with 1-year follow-ups. A post-treatment surgery survey and on-site examination were administered and patients' data from the clinical trial records and additional interview data were analyzed to evaluate the surgical intervention rate. Patients' lumbar disease symptoms, Oswestry Disability Index, and imaging features were evaluated. RESULTS: Among the patients (condoliase, n = 228; placebo, n = 128) enrolled in the clinical trials, additional post-treatment surgery data were available for 231 patients after the clinical trials ended, and 179 patients underwent post-trial examinations, at least 5 years and 17 months after the end of the clinical trials. The surgical intervention rate in the placebo and condoliase groups was 20.7% (95% confidence interval: 14.2-29.7) and 13.4% (95% confidence interval: 8.8-20.2), respectively. The mean change in Oswestry Disability Index score from pre-injection in placebo and condoliase groups was -24.7 ± 15.0 and -32.7 ± 18.6 (between-group difference: -8.0 ± 17.3; 95% confidence interval: -13.2 to -2.7). Modic Type 2 changes were observed, particularly in the condoliase group. No relationship between lumbar disease symptoms and change in imaging features was found. CONCLUSIONS: This follow-up study more than 1 year revealed no new safety concerns of condoliase. However, because the study had several limitations, such as large loss of follow-up, further research is needed.

Abrogation of LBX1 in skeletal muscle results in hypoplastic limbs and progressive kyphosis in mice.

LBX1 is a gene located near a single-nucleotide polymorphism, rs11190870, which is highly associated with susceptibility to adolescent idiopathic scoliosis. However, the potential involvement of LBX1 in the etiology of this spinal deformity has not been elucidated. In this study, we aimed to determine whether the lack of LBX1 in skeletal muscle results in spinal deformities in mice. We generated mutant mice in which the Lbx1 allele was conditionally excised under the control of a human muscle actin promoter. Mice lacking LBX1 from the skeletal muscle were fertile and available. The mutant mice had hypoplastic forelimbs and weighed less than control animals, but otherwise, there were no overt anomalies. The mice did not exhibit a scoliosis-like spinal deformity; however, they developed moderate kyphosis as they grew old. These observations indicated that LBX1 is involved in limb development and potentially in the maintenance of spinal curvature/alignment in mice.

Prevalence of low bone mineral density and risk of fractures in osteosarcoma and Ewing's sarcoma survivors: A scoping review.

Background: The clinical outcomes of patients with pediatric cancer have significantly improved over the past few decades. However, the treatments are often highly intensive and can advertently pose a risk for developing various health conditions, including bone mass loss and fragility fractures. Since patients with bone malignancies, such as osteosarcoma (OS) and Ewing's sarcoma (ES), require musculoskeletal surgery as well as chemotherapy, OS/ES survivors are potentially at even greater risk of developing these musculoskeletal conditions than those with other types of cancer. However, these issues in OS/ES survivors are often overlooked by clinicians treating childhood cancers. Thus, this scoping review was designed and conducted to better understand the bone health conditions in OS/ES survivors. Design: We conducted a literature search and included the studies that describe bone mineral density in association with bone health in OS/ES survivors for analysis. Data regarding patients' demographic, diagnosis, bone mineral density, laboratory examinations, and incidence of fractures were extracted and evaluated. Results: We found that almost half of OS/ES survivors have bone mass deficit and that several factors (such as a frailer physique and younger age at diagnosis) are potentially associated with low bone mass in OS/ES survivors. On the other hand, due to a paucity of information currently available, we could not determine whether long-term OS/ES survivors would ultimately regain bone mass or be at a greater risk of fragility fractures. Conclusions: This scoping review reveals a previously unappreciated knowledge gap in our understanding of bone health conditions in OS/ES survivors and raises awareness among clinicians and care providers of this condition that OS/ES patients may encounter after successful treatment.

Anti-Barnacle Activities of Isothiocyanates Derived from ß-Citronellol and Their Structure-Activity Relationships.

Antifouling agents with low toxicity are in high demand for sustaining marine industries and the environment. This study aimed to synthesize 15 isothiocyanates derived from ß-citronellol and evaluate their antifouling activities and toxicities against cypris larvae of the barnacle Amphibalanus amphitrite. The synthesized isothiocyanates exhibited effective antifouling activities (EC50 =0.10-3.33 µg mL-1 ) with high therapeutic ratios (LC50 /EC50 >30). Four isothiocyanates with an amide or isocyano group showed great potential as effective antifouling agents (EC50 =0.10-0.32 µg mL-1 , LC50 /EC50 =104-833). The enantiomers of the isothiocyanates only slightly differed in their antifouling activities. These results may serve as a basis for further research and development of ß-citronellol-derived isothiocyanates as effective low-toxic antifouling agents. To the best of our knowledge, this study is the first to report the antifouling activities of isothiocyanates derived from accessible natural products.

Identification of a Functional Susceptibility Variant for Adolescent Idiopathic Scoliosis that Upregulates Early Growth Response 1 (EGR1)-Mediated UNCX Expression.

Adolescent idiopathic scoliosis (AIS) is a serious health problem affecting 3% of live births all over the world. Many loci associated with AIS have been identified by previous genome wide association studies, but their biological implication remains mostly unclear. In this study, we evaluated the AIS-associated variants in the 7p22.3 locus by combining in silico, in vitro, and in vivo analyses. rs78148157 was located in an enhancer of UNCX, a homeobox gene and its risk allele upregulated the UNCX expression. A transcription factor, early growth response 1 (EGR1), transactivated the rs78148157-located enhancer and showed a higher binding affinity for the risk allele of rs78148157. Furthermore, zebrafish larvae with UNCX messenger RNA (mRNA) injection developed body curvature and defective neurogenesis in a dose-dependent manner. rs78148157 confers the genetic susceptibility to AIS by enhancing the EGR1-regulated UNCX expression. © 2022 American Society for Bone and Mineral Research (ASBMR).

Effect of contrast media on the enzyme activity of condoliase: In vitro assessment.

Background: Condoliase has been used in Japan to treat patients with lumbar disc herniation by its injection into the nucleus pulposus. The injection of condoliase together with contrast media is prohibited; because there are no data whether contrast media have any effect on condoliase activity. This study aimed to elucidate the effects of contrast media on condoliase activity. Methods: Condoliase with chondroitin sulfate (CS) and without CS were mixed with various contrast media (nonionic [iohexol or iotrolan]; ionic [amidotrizoic acid]). (i) The mixtures with CS were incubated at 37°C; (ii) the mixtures without CS were stored at 24°C for 60 min, followed by addition of CS to assess condoliase activity by measuring the amount of N-acetylhexosamines enzymatically cleaved from CS using Morgan-Elson method. Results: (i) In the presence of CS, the ionic contrast media reduced condoliase activity within 10 min in a dose-dependent manner, and the nonionic contrast media had no effect on condoliase activity for at least 120 min. (ii) In the absence of CS, the ionic contrast media almost completely inactivated condoliase within 15 min, and the nonionic contrast media also reduced condoliase activity; the residual activity was 65% with iotrolan and 35% with iohexol at 60 min. Conclusions: The ionic contrast media significantly reduced condoliase activity regardless of presence or absence of CS. Although the nonionic contrast media did not affect condoliase activity in the presence of CS, it reduced activity in the absence of CS. Mixing condoliase with contrast media, especially ionic type contrast media, should be avoided.

Oxidation Potential Gap (ΔEox ): The Hidden Parameter in Redox Chemistry.

Oxidative biaryl coupling of aryls with different electronic features generally fails. However, this has not been systematically studied via theoretical analysis, and thus, the crucial factor governing coupling efficiency remains unclear. Herein, we propose that the "oxidation potential gap (ΔEox )" is a key parameter in predicting the efficiency of an intramolecular oxidative coupling reaction, with ΔEox defined as a difference in the oxidation potentials of the relevant aromatic rings. Our experimental and computational analyses revealed that the efficiency of an aromatic intramolecular coupling reaction correlates with the activation energy (ΔE≠ ) of C-C bond formation of the radical cation intermediates. Furthermore, ΔE≠ correlates with ΔEox . Therefore, we demonstrate the tuning of ΔEox by attaching cleavable extra electron-donating/-withdrawing groups, enabling the rational synthesis of a phenanthridone skeleton using aromatic rings with an electronic gap.

Risk assessment of femoral pathological fracture in prostate cancer patients by computed tomography analysis.

INTRODUCTION: Prostate cancer often forms osteoblastic lesions that appear as a high-dense shadow upon X-ray. Although the lesions may seem to increase bone strength, pathological fracture occurs in one in four patients with prostate cancer. The aim of this study is to elucidate the factors that may increase the risk of pathological fracture in patients with prostate cancer metastases in the proximal femur by analyzing computed tomography data. MATERIALS AND METHODS: Computed tomography data of the femur of 62 prostate cancer patients were retrospectively analyzed. The patients were divided into three groups based on the presence or absence of femoral metastatic lesions and pathological fracture. Surgical specimens of the proximal femur collected from patients who had a pathological fracture were histologically analyzed. RESULTS: Bone density in the marrow area was increased in all cases with metastases compared with those with no metastases. Contrarily, the cortical bone density at the medial trochanter region was significantly lower in patients who had pathological fractures in the proximal femur than those who did not. Accordingly, histological analysis of the surgical specimens revealed that the affected cortical bone was osteopenic without any apparent new bone formation. CONCLUSION: These results indicate that prostate cancer is less effective in inducing bone formation in the cortex than in the marrow and that the decrease in the cortical bone density at the medial trochanter region leads to an increased risk of pathological fracture. Therefore, a previously undocumented risk factor for pathological fracture in prostate cancer patients is presented.

Spontaneous Osseous Fusion after Remodeling Therapy for Chronic Atlantoaxial Rotatory Fixation and Recovery Mechanism of Rotatory Range of Motion of the Cervical Spine.

We aimed to investigate the risk factors of spontaneous osseous fusion (SOF) of the atlantoaxial joint after closed reduction under general anesthesia followed by halo fixation (remodeling therapy) for chronic atlantoaxial rotatory fixation, and to elucidate the recovery mechanism of the rotatory range of motion (ROM) after halo removal. Twelve patients who underwent remodeling therapy were retrospectively reviewed. Five patients with SOF were categorized as the fusion group and seven patients without SOF as the non-fusion group. Three dimensional CT was used to detect direct osseous contact (DOC) of facet joints before and during halo fixation, while dynamic CT at neutral and maximally rotated head positions was performed to measure rotatory ROM after halo removal. The duration from onset to initial visit was significantly longer (3.2 vs. 5.7 months, p = 0.04), incidence of DOC during halo fixation was higher (0/7 [0%] vs. 4/5 [80%], p = 0.004), and segmental rotatory ROM of Occiput/C1 (Oc/C1) at final follow-up was larger (9.8 vs. 20.1 degrees, p = 0.003) in the fusion group. Long duration from the onset to the initial visit might induce irreversible damage to the articular surface of the affected facet, which was confirmed as DOC during halo fixation and resulted in SOF. Long duration from the onset to the initial visit and DOC during halo fixation could be used to suggest the risk for SOF. Nonetheless, rotatory ROM of Oc/C1 increased to compensate for SOF.

Increased migratory activity and cartilage regeneration by superficial-zone chondrocytes in enzymatically treated cartilage explants.

BACKGROUND: Limited chondrocyte migration and impaired cartilage-to-cartilage healing is a barrier in cartilage regenerative therapy. Collagenase treatment and delivery of a chemotactic agent may play a positive role in chondrocyte repopulation at the site of cartilage damage. This study evaluated chondrocyte migratory activity after enzymatic treatment in cultured cartilage explant. Differential effects of platelet-derived growth factor (PDGF) dimeric isoforms on the migratory activity were investigated to define major chemotactic factors for cartilage. METHODS: Full-thickness cartilage (4-mm3 blocks) were harvested from porcine femoral condyles and subjected to explant culture. After 15 min or 60 min of actinase and collagenase treatments, chondrocyte migration and infiltration into a 0.5-mm cartilage gap was investigated. Cell morphology and lubricin, keratan sulfate, and chondroitin 4 sulfate expression in superficial- and deep-zone chondrocytes were assessed. The chemotactic activities of PDGF-AA, -AB, and -BB were measured in each zone of chondrocytes, using a modified Boyden chamber assay. The protein and mRNA expression and histological localization of PDGF-ß were analyzed by western blot analysis, real-time reverse transcription polymerase chain reaction (RT-PCR), and immunohistochemistry, and results in each cartilage zone were compared. RESULTS: Superficial-zone chondrocytes had higher migratory activity than deep-zone chondrocytes and actively bridged the cartilage gap, while metachromatic staining by toluidine blue and immunoreactivities of keratan sulfate and chondroitin 4 sulfate were detected around the cells migrating from the superficial zone. These superficial-zone cells with weak immunoreactivity for lubricin tended to enter the cartilage gap and possessed higher migratory activity, while the deep-zone chondrocytes remained in the lacuna and exhibited less migratory activity. Among PDGF isoforms, PDGF-AB maximized the degree of chemotactic activity of superficial zone chondrocytes. Increased expression of PDGF receptor-ß was associated with higher migratory activity of the superficial-zone chondrocytes. CONCLUSIONS: In enzymatically treated cartilage explant culture, chondrocyte migration and infiltration into the cartilage gap was higher in the superficial zone than in the deep zone. Preferential expression of PDGF receptor-ß combined with the PDGF-AB dimeric isoform may explain the increased migratory activity of the superficial-zone chondrocytes. Cells migrating from superficial zone may contribute to cartilage regeneration.

Establishment of a suitable combination of serological markers to diagnose surgical site infection following spine surgery: A novel surgical site infection scoring system.

BACKGROUND: The accuracy rates of several effective serological markers of surgical site infection following spine surgery are unclear. We aimed to verify the accuracy of each significant marker and identify the most suitable and effective combination of these markers for the diagnosis of surgical site infection following spine surgery. METHODS: This retrospective study enrolled 329 patients who underwent spine surgery for causes other than infectious spondylitis, including 9 patients with surgical site infection. Complete blood cell count, differential counts, and C-reactive protein levels were measured preoperatively and postoperatively (days 2 and 7). Serological data were compared among non-surgical site infection and surgical site infection cases. Cutoff values for items presenting significant differences were determined using receiver operating characteristic curves. Ratios in each serological factor at each time-point were compared. Combinations of these factors on postoperative day 7 and ratio items were investigated to determine the most suitable combination comprising the least number of items. RESULTS: Significant differences were noted among four factors on postoperative day 7, except for the lymphocyte count. For the ratio items, significant differences were observed among 6 items. The combination of these ten markers was examined; each factor was assigned 1 point. The most suitable combination comprising 4 items, including neutrophil count, neutrophil-to-lymphocyte ratio, lymphocyte count ratio, and C-reactive protein ratio, presented an AUC of 0.95, with a cutoff value, sensitivity, and specificity of 3 points, 0.89, and 0.92, respectively. CONCLUSION: The combination of four markers is the most suitable criterion for the surgical site infection scoring system following spine surgery, where scores of ≥3 points strongly indicate surgical site infection. This criterion may be a strong tool for detecting surgical site infection.

A novel approach for identifying serological markers indicative of surgical-site infection following spine surgery: Postoperative lymphopenia is a risk factor.

BACKGROUND: Postoperative lymphopenia (PL) after spine surgery is reported to be an indicator of surgical-site infection (SSI). PL without SSI is often encountered, resulting in a treatment dilemma. We focused on PL, so as to improve the accuracy of detecting SSI. METHODS: In total, 329 patients underwent spine surgery, including nine patients presenting with SSI. The complete blood cell counts, differential counts, and C-reactive protein (CRP) level were measured pre-surgery and on postoperative days 2, 7, and 14. The relationships between PL and SSI were evaluated, and PL and non-PL conditions were compared among all cases. We then divided the patients into two groups: PL and non-PL, and determined the useful serological markers using receiver operating characteristic curves. RESULTS: Sixty-one patients presented with PL, including four with SSI. However, PL was not directly suggested as a biomarker of SSI (p = 0.067). We revealed PL as a risk factor for SSI (p = 0.004, Odds ratio: 7.54). Among all cases, the lymphocyte count and CRP level differed significantly between the PL and non-PL groups at all perioperative time-points. The white blood cell count, neutrophil count, and CRP levels on postoperative day 7 significantly differed between the SSI and non-SSI cases in the PL group. The area under the curve (AUC) for CRP was greater than that of the other parameters. The neutrophil count was only effective as a marker in the non-PL group. The combination of two cutoff values (CRP: 3.7 mg/dL (PL group) and neutrophil count: 6172/µL (non-PL group)) presented high specificity (87.2%) and sensitivity (88.9%), while only one cutoff value (CRP: 2.9 mg/dL) had a specificity of 77.4% and sensitivity of 77.8%. CONCLUSIONS: Approximately one-fifth of patients developed PL post-surgery, which was a risk factor for SSI, with constant high inflammation. Grouping based on PL and establishing diagnostic cutoff values are more appropriate than establishing only one cutoff value for overall cases.

Trabectedin suppresses osteosarcoma pulmonary metastasis in a mouse tumor xenograft model.

Osteosarcoma (OS) is the most common primary bone tumor that mainly affects adolescents and young adults. Although standard treatment modality can achieve up to 60%-70% 5-year survival rate, there has not been any substantial improvement over the past four decades. Furthermore, those presenting with pulmonary metastatic lesions often undergo a highly unfavorable clinical course. Therefore, there is a severely unmet clinical need to provide a more effective treatment for patients with OS. In this study, we show that trabectedin (TBD), a chemotherapeutic agent approved for soft tissue sarcomas, significantly suppresses pulmonary metastasis in a mouse OS xenograft model. In vitro experiments revealed that TBD suppresses cell migration potentially by downregulating the activity of ERK1/2, intracellular molecules that are critically involved in the regulation of cell motility. Collectively, our data may provide a basis for further investigation of TBD on the potential use for OS patients who are at great risk of pulmonary metastasis.