Serine Protease Inhibitor Kazal Type Research Articles

P442: SPINK2 PROTEIN EXPRESSION QUANTIFIED BY IMMUNOHISTOCHEMISTRY REFINES RISK STRATIFICATION AND PREDICTS THERAPY OUTCOMES IN AML

Background: Suboptimal prognostication, therapy refractoriness and relapse contribute to the poor outcome of Acute Myeloid Leukemia (AML) patients. Leukemic stem cells (LSC) are considered crucial drivers of relapse and therapy resistance. Clinicopathological characterization of LSC-associated genes with convenient assessment tools such as immunohistochemistry (IHC) is needed. Our initial screening of AML datasets for LSC-associated genes identified Serine Protease Inhibitor Kazal type 2 (SPINK2) with high expression in AML, particularly in LSC fractions. However, in-depth analysis of its clinicopathological associations and prognostic utility in AML are lacking. Aims: To assess SPINK2 expression in adult AML with IHC, and determine its clinicopathological associations, prognostic impact and association with therapy response Methods: We studied the expression of SPINK2 by IHC in 172 non-M3 adult AML patients (median age:52yrs, range: 18-86yrs) treated at the Prince of Wales Hospital. The majority (90.8%) were de novo type and 72.3% had intermediate-risk(IR) cytogenetics, with 88.5% receiving Daunorubicin + Ara-C (DA 7 + 3) induction. SPINK2 expression was assessed by 3 hematopathologists in a double-blinded manner and quantified by a composite score (range: 0-16). Clinical data was collected for all cases. Results:SPINK2 staining in leukemic blasts was consistently cytoplasmic, and its protein expression strongly correlated with mRNA levels by qPCR in a subset of 128 patients (r=0.716, P<0.0001). The cohort was dichotomized at the median score 3, since this cut-off showed strongest association with poor event-free survival (EFS) and overall survival (OS). High SPINK2 (SPINK2hi) patients accounted for 77/172 (44.8%) cases, and low SPINK2 (SPINK2lo) patients for 95/172(55.2%). SPINK2hi associated with IR cytogenetics (P=0.014), normal karyotype (P=0.019) and NPM1 mutation (P<0.001), while SPINK2lo with t(8;21) and CEBPA double-mutation (both P<0.001). Survival and treatment-response analyses were performed on a subgroup of 137 patients who received DA 7 + 3 based induction. SPINK2hi patients had lower CR rates (73.8% vs 88.3%,P=0.028), higher 6-month relapse rates (31.8% vs 9.1%,P=0.004), lower 5-yr RFS (25.8% vs 46.8%,P=0.004), EFS (16.6% vs 37.2%,P<0.001) and OS (25.3% vs 51.2%,P<0.001). SPINK2 status also identified high risk patients of the IR cytogenetic subgroup. Of these, SPINK2hi patients had lower CR rates (68.6% vs 90.0%,P=0.013), higher 6-month relapse rates (31.4% vs 6.9%,P=0.018), lower 5-yr RFS(27.0% vs 44.6%,P=0.018), EFS(15.4% vs 38.3%,P<0.001) and OS(22.9% vs 51.5%,P=0.002). In this subgroup, median SPINK2 score was higher in patients requiring ≥2 inductions to achieve CR vs. patients requiring 1 course (7 vs 2,P=0.009). Multivariate analyses in whole and IR cytogenetics cohorts showed the poor prognostic effect of SPINK2hi on EFS and OS independent of age, cytogenetic risk, mutations and achievement of CR1, including stem cell transplantation given in CR (Table 1). In our NPM1mut subgroup (N=41), SPINK2hi status associated with poor EFS (HR:3.3,P=0.04) and OS (HR: 4.0,P=0.007) independent of age and FLT3-ITD with high allelic ratio. Incorporation of SPINK2 with FLT3 status refined ELN2017 risk definition, whereby 15/22 favorable-risk patients could be reclassified as intermediate-risk. Image:Summary/Conclusion: High SPINK2 protein expression by IHC predicted increased chemoresistance and early relapse risk in AML, and was an independent adverse prognostic biomarker, particularly in patients with intermediate-risk cytogenetics and NPM1 mutations.

Role of endoscopic ultrasound and cyst fluid tumor markers in diagnosis of pancreatic cystic lesions.

BACKGROUNDPancreatic cystic lesions (PCLs) are common in clinical practice. The accurate classification and diagnosis of these lesions are crucial to avoid unnecessary treatment of benign lesions and missed opportunities for early treatment of potentially malignant lesions. AIMTo evaluate the role of cyst fluid analysis of different tumor markers such as cancer antigens [e.g., cancer antigen (CA)19-9, CA72-4], carcinoembryonic antigen (CEA), serine protease inhibitor Kazal-type 1 (SPINK1), interleukin 1 beta (IL1-β), vascular endothelial growth factor A (VEGF-A), and prostaglandin E2 (PGE2)], amylase, and mucin stain in diagnosing pancreatic cysts and differentiating malignant from benign lesions. METHODSThis study included 76 patients diagnosed with PCLs using different imaging modalities. All patients underwent endoscopic ultrasound (EUS) and EUS-fine needle aspiration (EUS-FNA) for characterization and sampling of different PCLs. RESULTSThe mean age of studied patients was 47.4 ± 11.4 years, with a slight female predominance (59.2%). Mucin stain showed high statistical significance in predicting malignancy with a sensitivity of 87.1% and specificity of 95.56%. It also showed a positive predictive value and negative predictive value of 93.1% and 91.49%, respectively (P < 0.001). We found that positive mucin stain, cyst fluid glucose, SPINK1, amylase, and CEA levels had high statistical significance (P < 0.0001). In contrast, IL-1β, CA 72-4, VEGF-A, VEGFR2, and PGE2 did not show any statistical significance. Univariate regression analysis for prediction of malignancy in PCLs showed a statistically significant positive correlation with mural nodules, lymph nodes, cyst diameter, mucin stain, and cyst fluid CEA. Meanwhile, logistic multivariable regression analysis proved that mural nodules, mucin stain, and SPINK1 were independent predictors of malignancy in cystic pancreatic lesions. CONCLUSIONEUS examination of cyst morphology with cytopathological analysis and cyst fluid analysis could improve the differentiation between malignant and benign pancreatic cysts. Also, CEA, glucose, and SPINK1 could be used as promising markers to predict malignant pancreatic cysts.

Combined SPINK1 mutations induce early-onset severe chronic pancreatitis in a child with severe obesity.

SummaryThe most frequent causes of pancreatitis classically have been known to be gallstones or alcohol. However, genetics can also play a key role in predisposing patients to both chronic and acute pancreatitis. The serine protease inhibitor Kazal type 1 (SPINK 1) gene is known to be strongly associated with pancreatitis. Patients with these underlying genetic mutations can have severe diseases with a high morbidity rate and frequent hospitalization. We report an Arab girl who presented with acute pancreatitis at the age of 7 years progressing to recurrent chronic pancreatitis over a few years. She had severe obesity from the age of 4 years and developed type 2 diabetes at the age of 12. She had a normal biliary system anatomy. Genetic analysis showed that she had combined heterozygous mutations in the SPINK1 gene (SPINK1, c.101A>G p.(Asn34Ser) and SPINK1, c.56-37T>C). Her parents were first-degree cousins, but neither had obesity. Mother was detected to have the same mutations. She had type 2 diabetes but never presented with pancreatitis. This case is the first to be reported from the Arab region with these combined mutations leading to recurrent chronic pancreatitis. It illustrates the importance of diagnosing the underlying genetic mutation in the absence of other known causes of pancreatitis. Considering the absence of pancreatitis history in the mother who did not have obesity but harboured the same mutations, we point out that severe obesity might be a triggering factor of pancreatitis in the presence of the mutations in SPINK1 gene in this child. While this is not an assumption from a single patient, we show that not all carriers of this mutation develop the disease even within the same family. Triggering factors like severe obesity might have a role in developing the disease.Learning pointsAcute recurrent pancreatitis and chronic pancreatitis are uncommon in children but might be underdiagnosed.Biliary tract anomalies and dyslipidaemias are known causative factors for pancreatitis, but pancreatitis can be seen in children with intact biliary system.Genetic diagnosis should be sought in children with pancreatitis in the absence of known underlying predisposing factors.SPINK1 mutations can predispose to an early-onset severe recurrent pancreatitis and acute pancreatitis.

A Kazal-type serine protease inhibitor mediates innate immunity in wild silkworm Actias selene Hübner

The prophenoloxidase (PPO) activation system is an important innate immune defense mechanism in arthropods. Actias selene is a rare and important wild silk insect that can spin high-quality cocoon silk, but, other than its morphology, its molecular mechanism is rarely reported. Here, we report the purification and characterization of a novel KSPI gene from A. selene (AsKSPI, which can negatively regulate PPO activation. Its open reading frame (ORF) was 291 bp, encoding 96 amino acids. Real-time quantitative PCR (RT-qPCR) showed that AsKSPI mRNA was significantly expressed in the fat body. Immunostimulatory tests showed that the mRNA levels of AsKSPI in the fat body were up-regulated following injection of Micrococcus luteus, Escherichia coli, Beauveria bassiana, and nuclear polyhedrosis virus (NPV). Enzyme activity experiments showed that the purified recombinant AsKSPI could inhibit the activation of PPO in hemolymph of A. selene, but did not affect phenoloxidase (PO) activity after PPO had been activated. So, AsKSPI could regulate the innate immunity of A. selene through the PPO cascade. These findings will contribute to the understanding of the immune mechanism of wild silkworm and provide a basis for better protection and utilization of special economic insect resources.

How, where and when is SPINK3 bound and removed from mouse sperm?

Sperm capacitation in mammals is a fundamental requirement to acquire their fertilizing capacity. Little is known about the action mechanism of the molecules that prevent capacitation from occurring prematurely. These molecules are known as decapacitation factors (DFs) and they must be removed from the sperm surface for capacitation to occur successfully. Serine protease inhibitor Kazal type 3 (SPINK3) has been proposed as one of these DFs. Here, we evaluate how this protein binds to mouse sperm and its removal kinetics. We describe that SPINK3 is capable of binding to the membrane of mature epididymal sperm through protein-lipid interactions, specifically to lipid rafts subcellular fraction. Moreover, cholera toxin subunit b (CTB) avoids SPINK3 binding. We observe that SPINK3 is removed from the sperm under in vitro capacitating conditions and by the uterine fluid from estrus females. Our ex vivo studies show the removal kinetics of this protein within the female tract, losing SPINK3 formerly from the apical region of the sperm in the uterus and later from the flagellar region within the oviduct. The presence of acrosome-reacted sperm in the female duct concurs with the absence of SPINK3 over its surface.

Diagnostic testing of eczematous dermatitis with incomplete response to dupilumab

Diagnostic testing of eczematous dermatitis with incomplete response to dupilumab

Structural and Biophysical Insights into SPINK1 Bound to Human Cationic Trypsin.

(1) The serine protease inhibitor Kazal type 1 (SPINK1) inhibits trypsin activity in zymogen granules of pancreatic acinar cells. Several mutations in the SPINK1 gene are associated with acute recurrent pancreatitis (ARP) and chronic pancreatitis (CP). The most common variant is SPINK1 p.N34S. Although this mutation was identified two decades ago, the mechanism of action has remained elusive. (2) SPINK1 and human cationic trypsin (TRY1) were expressed in E. coli, and inhibitory activities were determined. Crystals of SPINK1–TRY1 complexes were grown by using the hanging-drop method, and phases were solved by molecular replacement. (3) Both SPINK1 variants show similar inhibitory behavior toward TRY1. The crystal structures are almost identical, with minor differences in the mutated loop. Both complexes show an unexpected rotamer conformation of the His63 residue in TRY1, which is a member of the catalytic triad. (4) The SPINK1 p.N34S mutation does not affect the inhibitory behavior or the overall structure of the protein. Therefore, the pathophysiological mechanism of action of the p.N34S variant cannot be explained mechanistically or structurally at the protein level. The observed histidine conformation is part of a mechanism for SPINK1 that can explain the exceptional proteolytic stability of this inhibitor.

Regulatory effects of Trichinella spiralis and a serine protease inhibitor on the endoplasmic reticulum stress response of intestinal epithelial cells

The accumulation of unfolded or misfolded proteins in the endoplasmic reticulum can cause an endoplasmic reticulum stress (ERS) response. If ERS continues or cannot be alleviated, it will cause the production of proapoptotic factors and eventually lead to apoptosis. Therefore, this study mainly explored whether Trichinella spiralis Kazal-type serine protease inhibitor (TsKaSPI) contributed to the invasion of intestinal epithelial cells during the infectious stage of T. spiralis by regulating ERS. First, in the T. spiralis infection model, H&E staining was used to analyse the damage to jejunum tissue, a TUNEL assay was used to examine cell apoptosis, and the expression of ERS-related and apoptosis-related molecules was also measured. The results showed that ERS occurred during the intestinal phase of T. spiralis infection, while remission began during the cyclic phase. Then, we selected TsKaSPI, one of the important components of T. spiralis ES antigens, for in vitro experiments. The results showed that TsKaSPI could induce apoptosis in a porcine small intestinal epithelial cell line (IPEC cells) by activating ERS and promote activation of the NF-κB signalling pathway. Inhibition experiments confirmed that the occurrence of ERS was accompanied by the activation of NF-κB, and the two processes regulated each other. Finally, we conducted in vivo experiments and administered TsKaSPI to mice. The results confirmed that TsKaSPI could activate ERS and lead to apoptosis in intestinal epithelial cells. In conclusion, T. spiralis infection and TsKaSPI can promote cell apoptosis by activating the ERS response in intestinal epithelial cells and activate the NF-κB signalling pathway to promote the occurrence and development of inflammation.

Raised SPINK1 levels play a role in angiogenesis and the transendothelial migration of ALL cells

The present study was designed to assess whether raised Serine protease inhibitor Kazal type 1 (SPINK1) expressions modulates angiogenesis. Human umbilical vein endothelial cells (HUVECs) exposed to SPINK1 were noted to exhibit raised expressions of interleukin-8 (IL-8) as well as VCAM-1 and ICAM-1 cell adhesion molecules in a dose-dependent manner. In co-culture system of HUVECs and Acute lymphoblastic leukemia (ALL) cells, SPINK1 exposure also resulted in enhanced endothelial cell motility and ALL cells trans-endothelial migration. High concentrations of SPINK1 caused in vitro cellular reorganization into tubes in Matrigel-cultured HUVECs and induced in vivo vascularization and brain infiltration of NOD/SCID ALL model mice. The further transcriptomic analysis indicated that SPINK1 treatment altered several biological processes of endothelial cells and led to activation of the MAPK pathway. This study is the first to determine the neovascularization effects of raised SPINK1.

Collision tumors revealed by prospectively assessing subtype-defining molecular alterations in 904 individual prostate cancer foci

BACKGROUNDProstate cancer is multifocal with distinct molecular subtypes. The utility of genomic subtyping has been challenged due to inter- and intrafocal heterogeneity. We sought to characterize the subtype-defining molecular alterations of primary prostate cancer across all tumor foci within radical prostatectomy (RP) specimens and determine the prevalence of collision tumors.METHODSFrom the Early Detection Research Network cohort, we identified 333 prospectively collected RPs from 2010 to 2014 and assessed ETS-related gene (ERG), serine peptidase inhibitor Kazal type 1 (SPINK1), phosphatase and tensin homolog (PTEN), and speckle type BTB/POZ protein (SPOP) molecular status. We utilized dual ERG/SPINK1 immunohistochemistry and fluorescence in situ hybridization to confirm ERG rearrangements and characterize PTEN deletion, as well as high-resolution melting curve analysis and Sanger sequencing to determine SPOP mutation status.RESULTSBased on index focus alone, ERG, SPINK1, PTEN, and SPOP alterations were identified in 47.5%, 10.8%, 14.3%, and 5.1% of RP specimens, respectively. In 233 multifocal RPs with ERG/SPINK1 status in all foci, 139 (59.7%) had discordant molecular alterations between foci. Collision tumors, as defined by discrepant ERG/SPINK1 status within a single focus, were identified in 29 (9.4%) RP specimens.CONCLUSIONInterfocal molecular heterogeneity was identified in about 60% of multifocal RP specimens, and collision tumors were present in about 10%. We present this phenomenon as a model for the intrafocal heterogeneity observed in previous studies and propose that future genomic studies screen for collision tumors to better characterize molecular heterogeneity.FUNDINGEarly Detection Research Network US National Cancer Institute (NCI) 5U01 CA111275-09, Center for Translational Pathology at Weill Cornell Medicine (WCM) Department of Pathology and Laboratory Medicine, US NCI (WCM SPORE in Prostate Cancer, P50CA211024-01), R37CA215040, Damon Runyon Cancer Research Foundation, US MetLife Foundation Family Clinical Investigator Award, Norwegian Cancer Society (grant 208197), and South-Eastern Norway Regional Health Authority (grant 2019016 and 2020063).

Nuclear magnetic resonance spectroscopy reveals dysregulation of monounsaturated fatty acid metabolism upon SPINK1 attenuation in colorectal cancer.

Metabolic reprogramming, a key hallmark of cancer, plays a pivotal role in fulfilling the accelerated biological demands of tumor cells. Such metabolic changes trigger the production of several proinflammatory factors, thereby inciting cancer development and its progression. Serine protease inhibitor Kazal Type 1 (SPINK1), well known for its oncogenic role and its upregulation via acute-phase reactions, is highly expressed in multiple cancers including colorectal cancer (CRC). Here, we show accumulation of lipid droplets in CRC cells stained with Oil Red O upon SPINK1 silencing. Furthermore, NMR spectroscopy analysis revealed an accretion of monounsaturated fatty acids (MUFAs) and phosphatidylcholine in these CRC cells, while the levels of polyunsaturated fatty acids remained unaltered. This alteration indicates the presence of MUFAs with the triglycerides in the lipid droplets as observed in SPINK1-silenced CRC cells. Considering the role of MUFAs in the anti-inflammatory response, our data hint that suppression of SPINK1 in CRC leads to activation of an anti-inflammatory signaling milieu. Conclusively, our study uncovers a connection between lipid metabolism and SPINK1-mediated CRC progression, hence paving the way for further exploration and better prognosis of SPINK1-positive CRC patients.

A Combined CRISP3 and SPINK1 Prognostic Grade in EPS-Urine and Establishment of Models to Predict Prognosis of Patients With Prostate Cancer.

BackgroundProstate cancer (PCa) is characterized by significant heterogeneity. Thus, novel prognostic indicators are required to improve prognosis and treatment.MethodsCysteine rich secretory protein 3 (CRISP3) and serine peptidase inhibitor Kazal type 1 (SPINK1) levels in expressed prostatic secretion (EPS)-urine collected during digital rectal examination of 496 patients histologically diagnosed with PCa were detected via enzyme-linked immunosorbent assay. A combined CRISP3 and SPINK1 prognostic grade (CSPG) was defined using cut-off values from receiver operating characteristic curves. Log-rank Kaplan-Meier survival curves investigated differences in prognosis between groups. Univariate and multivariate Cox analyses investigated the CSPG relationship with biochemical recurrence (BCR), cancer-specific survival (CSS), and overall survival (OS). Three prognostic models were developed and validated.ConclusionsCRISP3 and SPINK1 levels increased with Gleason score progression, pathological T stage, and metastasis status. CSPG in EPS-urine, which was an effective independent prognostic variable, accurately predicted the prognosis of patients with PCa. Three clinical prognostic models using the CSPG for BCR, CSS, and OS were developed and validated.

SPINKs in Tumors: Potential Therapeutic Targets

The serine protease inhibitor Kazal type (SPINK) family includes SPINK1-14 and is the largest branch in the serine protease inhibitor family. SPINKs play an important role in pancreatic physiology and disease, sperm maturation and capacitation, Nager syndrome, inflammation and the skin barrier. Evidence shows that the unregulated expression of SPINK1, 2, 4, 5, 6, 7, and 13 is closely related to human tumors. Different SPINKs exhibit various regulatory modes in different tumors and can be used as tumor prognostic markers. This article reviews the role of SPINK1, 2, 4, 5, 6, 7, and 13 in different human cancer processes and helps to identify new cancer treatment targets.

Value of pancreatic cyst fluid SPINK1 and glucose in differentiating potentially malignant cysts from those of benign nature: A prospective cohort study.

Diagnosis of malignant pancreatic cystic lesions (PCLs) is challenging as there is no investigation that offers both high diagnostic sensitivity and specificity for a definite diagnosis. Accurate diagnosis of cyst type is vital in order to not miss opportunities for early treatment of potentially malignant lesions and to avoid unnecessary surgeries. Serine protease inhibitor Kazal type I (SPINK1) and glucose are promising cyst fluid markers for differentiation of mucinous from non-mucinous cysts. We aim to validate the value of SPINK1 and glucose in detecting potentially malignant PCLs. A prospective study was conducted on 80 patients presenting with PCLs. Endoscopic ultrasound (EUS) evaluation of detailed cyst morphology and EUS with fine needle aspiration (FNA) were done. Fluid analysis for carcinoembryonic antigen (CEA), glucose and SPINK1 and cytopathology were done. We compared these data with the final diagnosis based on cytopathological and postoperative histopathological examination. Cyst fluid SPINK1 was significantly higher in malignant or potentially malignant cysts compared to benign cysts (0.91 vs 0.47 ng/ml; P = 0.001). Also, glucose was significantly lower in malignant or potentially malignant cysts compared to benign cysts (21.5 vs 68.5 mg/dl; P = 0.0001). Glucose and SPINK1 had the best sensitivity and specificity for differentiating mucinous from non-mucinous cysts with 84.78% and 73.53% (AUC 0.76; 95% CI [0.65-0.88]; cutoff value = 42 mg/dl), and 70.59% and 65.22% (AUC 0.72; 95% CI [0.64-0.86]; cutoff value = 0.58 ug/L) respectively. CEA level >192 ng/ml, high SPINK1 level and lymph node enlargement were the independent predictors of malignant cysts. Cyst fluid SPINK1 and glucose are promising diagnostic markers for the diagnosis of potentially malignant PCLs.

Hereditary Pancreatitis Related to SPINK-1 Mutation. Is There an Increased Risk of Developing Pancreatic Cancer?

It is thought that many of the idiopathic pancreatitis could have a genetic base.Approximately 50% of them correspond to CFTR (cystic fibrosis transmembrane conductance regulator gene) and SPINK-1 (serine protease inhibitor Kazal type 1) mutations.A recent study compares patients with acute pancreatitis andSPINK-1 mutation withpatients with idiopathic acute pancreatitis. The study highlights a12-fold increased risk of developing pancreatic cancer with SPINK-1 mutation versus the control group.Nonetheless, authors conclude that only specificpN34s mutation is related to pancreatic cancer. This relation is controversial, and international consensus guidelines for the follow-up in chronic pancreatitis with pancreatic cancer still do not recommend follow-up in SPINK-1 p. N34S mutation. We believe that developing prospective studies in which subgroups of patients with SPINK-1 mutation benefit from closer follow-ups would be necessary.

Similarities in DSG1 and KRT3 Downregulation through Retinoic Acid Treatment and PAX6 Knockdown Related Expression Profiles: Does PAX6 Affect RA Signaling in Limbal Epithelial Cells?

Congenital PAX6-aniridia is a rare panocular disease resulting from limbal stem cell deficiency. In PAX6-aniridia, the downregulation of the retinol-metabolizing enzymes ADH7 (All-trans-retinol dehydrogenase 7) and ALDH1A1/A3 (Retinal dehydrogenase 1, Aldehyde dehydrogenase family 1 member A3) have been described in limbal epithelial cells (LECs) and conjunctival epithelial cells. The aim of this study was to identify the role of retinol derivates in the differentiation of human LEC and its potential impact on aniridia-associated keratopathy development. Human LEC were isolated from healthy donor corneas and were cultured with retinol, retinoic acid, or pan-retinoic acid receptor antagonist (AGN 193109) acting on RARα, β, γ (NR1B1, NR1B2 NR1B3) or were cultured with pan-retinoid X receptor antagonist (UVI 3003) acting on RXR α, β, γ (retinoid X receptor, NR2B1, NR2B2, BR2B3). Using qPCR, differentiation marker and retinoid-/fatty acid metabolism-related mRNA expression was analysed. DSG1 (Desmoglein 1), KRT3 (Keratin 3), and SPINK7 (Serine Peptidase Inhibitor Kazal Type 7) mRNA expression was downregulated when retinoid derivates were used. AGN 193109 treatment led to the upregulation of ADH7, KRT3, and DSG1 mRNA expression and to the downregulation of KRT12 (Keratin 12) and KRT19 (Keratin 19) mRNA expression. Retinol and all-trans retinoic acid affect some transcripts of corneal LEC in a similar way to what has been observed in the LEC of PAX6-aniridia patients with the altered expression of differentiation markers. An elevated concentration of retinol derivatives in LEC or an altered response to retinoids may contribute to this pattern. These initial findings help to explain ocular surface epithelia differentiation disorders in PAX6-aniridia and should be investigated in patient cells or in cell models in the future in more detail.

SPINK1 as a plasma marker for tumor hypoxia and a therapeutic target for radiosensitization.

Hypoxia is associated with tumor radioresistance; therefore, a predictive marker for tumor hypoxia and a rational target to overcome it have been sought to realize personalized radiotherapy. Here, we show that serine protease inhibitor Kazal type I (SPINK1) meets these 2 criteria. SPINK1 expression was induced upon hypoxia (O2 < 0.1%) at the transcription initiation level in a HIF-dependent manner, causing an increase in secreted SPINK1 levels. SPINK1 proteins were detected both within and around hypoxic regions of xenografted and clinical tumor tissues, and their plasma levels increased in response to decreased oxygen supply to xenografts. Secreted SPINK1 proteins enhanced radioresistance of cancer cells even under normoxic conditions in EGFR-dependent and nuclear factor erythroid 2–related factor 2–dependent (Nrf2-dependent) manners and accelerated tumor growth after radiotherapy. An anti-SPINK1 neutralizing antibody exhibited a radiosensitizing effect. These results suggest that SPINK1 secreted from hypoxic cells protects the surrounding and relatively oxygenated cancer cells from radiation in a paracrine manner, justifying the use of SPINK1 as a target for radiosensitization and a plasma marker for predicting tumor hypoxia.

A Novel SPINK5 Gene Mutation Associated with Netherton Syndrome in an Omani Patient

Netherton syndrome (NS) is an autosomal recessive primary immunodeficiency. It is characterised by substantial skin barrier defects and is often misdiagnosed as severe atopic dermatitis or hyper-immunoglobulin E syndrome. Although more than 80 NS-associated pathogenic mutations in the serine peptidase inhibitor kazal type 5 (SPINK5) gene have been reported worldwide, only one has been reported in the Arab population to date. We report the case of a novel association between the c.1887+1G>A mutation in the SPINK5 gene and NS in an Omani-Arab patient born in 2014 who was managed at a paediatric immunology clinic in Muscat, Oman. Accurate genetic diagnosis facilitated tailored clinical management of the index patient and enabled the provision of genetic counselling and offering of future reproductive options to the individuals related to the index patient.

Protective effect of oligosaccharides isolated from Panax ginseng C. A. Meyer against UVB-induced skin barrier damage in BALB/c hairless mice and human keratinocytes

Ethnopharmacological relevanceSkin barrier dysfunction can lead to water and electrolyte loss, triggering homeostatic imbalances that can trigger atopic dermatitis and anaphylaxis. Panax ginseng C.A. Meyer is a traditional Chinese medicinal herb with known therapeutic benefits for the treatment of skin diseases, including photodamage repair effects and reduction of pigmentation. However, few reports exist that describe effectiveness of ginseng active components for repair of skin barrier damage. Materials and methodsGinseng oligosaccharide extract (GSO) was prepared from P. ginseng via water extraction followed by ethanol precipitation and resin and gel purification. GSO composition and structural characteristics were determined using LC-MS, HPLC, FT-IR, and NMR. To evaluate GSO as a skin barrier repair-promoting treatment, skin of UVB-irradiated BALB/c hairless mice was treated with or without GSO then skin samples were evaluated for epidermal thickness, transepidermal water loss (TEWL), and stratum corneum water content. In addition, UVB-exposed skin samples and HaCaT cells were analyzed to assess GSO treatment effects on levels of epidermal cornified envelope (CE) protein and other skin barrier proteins, such as filaggrin (FLG), involucrin (IVL), and aquaporin-3 (AQP3). Meanwhile, GSO treatment was also evaluated for effects on UVB-irradiated hairless mouse skin and HaCaT cells based on levels of serine protease inhibitor Kazal type-5 (SPINK5), trypsin-like kallikrein-related peptidase 5 (KLK5), chymotrypsin-like KLK7, and desmoglein 1 (DSG1). These proteins are associated with UVB-induced skin barrier damage manifesting as dryness and desquamation. ResultsGSO was shown to consist of oligosaccharides comprised of seven distinct types of monosaccharides with molecular weights of approximately 1 kDa that were covalently linked together via β-glycosidic bonds. In vivo, GSO applied to dorsal skin of BALB/c hairless mice attenuated UVB-induced epidermal thickening and moisture loss. Furthermore, GSO ameliorated UVB-induced reductions of levels of FLG, IVL, and AQP3 proteins. Additionally, GSO treatment led to increased DSG1 protein levels due to decreased expression of KLK7. In vitro, GSO treatment of UVB-irradiated HaCaT cells led to increases of FLG, IVL, and AQP3 mRNA levels and corresponding proteins, while mRNA levels of desquamation-related proteins SPINK5, KLK5, KLK7, and DSG1 and associated protein levels were restored to normal levels. ConclusionA P. ginseng oligosaccharide preparation repaired UVB-induced skin barrier damage by alleviating skin dryness and desquamation symptoms, highlighting its potential as a natural cosmetic additive that can promote skin barrier repair after UVB exposure.

CircRPS16 Promotes Proliferation and Invasion of Hepatocellular Carcinoma by Sponging miR-876-5p to Upregulate SPINK1.

The roles of serine protease inhibitor Kazal type 1 (SPINK1) in multiple types of cancers have been significantly documented. However, its specific roles in hepatocellular carcinoma (HCC) remain to be investigated. This study found that SPINK1 is upregulated in HCC and its upregulation correlates with poor prognosis. Besides, functional assays revealed that SPINK1 promotes cell proliferation, cell cycle, and invasion in vitro. Through bioinformatics analysis, we speculate that circRPS16 regulates SPINK1 expression by sponging miR-876-5p. This was further verified by the dual-luciferase reporter and fluorescent in situ hybridization (FISH) assays. Subsequently, rescue assays verified that circRPS16 promotes cell proliferation, cell cycle, and invasion through miR-876-5p. Importantly, silencing circRPS16 inhibited tumor growth by downregulating SPINK1 expression in vivo. Collectively, our results confirm that SPINK1 is a downstream target of circRPS16. Besides, circRPS16 and SPINK1 are oncogenic factors in HCC progression; they provide novel diagnostic and therapeutic targets for HCC patients.