SPRTN-dependent DPC degradation precedes repair of damaged DNA: a proof of concept revealed by the STAR assay.

DNA-protein crosslinks (DPCs), formed by the covalent conjugation of proteins to DNA, are toxic lesions that interfere with DNA metabolic processing and transcription. The development of an accurate biochemical assay for DPC isolation is a priority for the mechanistic understanding of their repair. Here, we propose the STAR assay for the direct quantification of DPCs, sensitive to physiologically relevant treatment conditions. Implementing the STAR assay revealed the formation of small cross-linked peptides on DNA, created by the proteolytic degradation of DPCs by SPRTN. The initial proteolytic degradation of DPCs is required for the downstream activation of DNA repair, which is mediated through the phosphorylation of H2Ax. This leads to the accumulation of DNA repair factors on chromatin and the subsequent complete removal of the cross-linked peptides. These results confirmed that the repair of DPCs is a two-step process, starting with proteolytic resection by SPRTN, followed by the repair of the underlying damage to the DNA.

Direct and cost-effective method for histone isolation from cultured mammalian cells.

Histones are an essential part of nucleosomes that regulate chromatin structure and function. Histone exchanges and modifications represent a scaffold for DNA transcription, repair, and replication. Studying histones and histone code is an important and fast-developing branch of epigenetic science. Here we propose a fast, efficient, and versatile assay for nucleosomal histone isolation from mammalian cells, without the use of acids or high salt solutions which are common for other histone isolation techniques. All components used in the protocol are common and inexpensive laboratory chemicals. The protocol has been evaluated on six commonly used cell lines and two animal tissue samples. The mild extraction conditions preserve delicate histone epigenetic changes, allowing its downstream analyses. We have demonstrated the assays' successful application during changes in the transcriptional activity of histone genes, cell cycle transitions, and DNA-damaging conditions. Histone fractions, obtained by the protocol, can be used for further applications, such as electrophoresis, immunoblot, and mass spectrometry. Therefore, the new proposed nucleosomal histone isolation method is sensitive, specific, and suitable for downstream applications of various kinds.

High NF-κB and STAT3 activity in human urothelial carcinoma: a pilot study.

PURPOSE: Given that the tumor-promoting inflammation has been previously established in squamous cell carcinoma of the bladder but its contribution to development of urothelial carcinoma (UC) still remains elusive, our aim was to study changes in expression and activity of inflammation-mediating NF-κB and STAT3 transcription factors in human urothelial bladder carcinoma as well as expression of their target genes cyclin D1, VEGFA and TGFß1. METHODS: Gene expression of STAT3, NF-κB, TGFß1, cyclin D1 and VEGFA was measured by quantitative real-time polymerase chain reaction in both tumor and healthy bladder tissue from 36 patients with UC of the bladder. Activation of STAT3 and NF-κB was assessed with immunohistochemistry and immunoblot. RESULTS: Urothelial bladder carcinoma displayed elevated expression as well as activation of NF-κB (P = 5.38e-10) and STAT3 (P = 0.002) transcription factors. Furthermore, elevated level of expression was observed for cyclin D1, VEGFA and TGFß1 (P = 9.71e-09, P = 9.71e-09, P = 5.38e-10). Preliminary statistical analysis indicated that the level of upregulation of STAT3 or NF-κB was probably not dependent upon the grade (P = 0.984 and 0.803, respectively) and invasiveness of the tumor (0.399 and 0.949), nor to the gender (0.780 and 0.536) and age (0.660 and 0.816) of the patients. CONCLUSIONS: NF-κB and STAT3 signaling pathways, as main inflammatory mediators, are found to be activated in urothelial bladder carcinoma indicating that chronic inflammatory processes are accompanying development of this tumor type. Future studies will have to determine possible causative role of inflammatory processes in development of urothelial bladder carcinomas.

SPRTN is involved in hepatocellular carcinoma development through the ER stress response.

Endoplasmic reticulum (ER) stress, prompted by the accumulation of misfolded or unfolded proteins, triggers the activation of the unfolded protein response (UPR) pathway to restore ER homeostasis. This stress response is implicated in the development of hepatocellular carcinoma (HCC). A biallelic mutation in SPRTN is currently the only known single-gene mutation implicated in the early onset of HCC. However, the exact mechanism linking SPRTN mutations to HCC remains unclear. In our study, we analyzed SPRTN and UPR in 21 human HCC tissue samples using RT-qPCR, immunoblot, and immunohistochemistry. We found alterations in the expression levels of SPRTN and UPR-related genes and proteins in HCC samples. The impact of SPRTN on the ER stress response was assessed in SPRTN-depleted HepG2 cells through RNA sequencing, pull-down assay, comet assay, and mitotic index calculation. We demonstrated that SPRTN interacts with the UPR sensor GRP78. Furthermore, we observed a decrease in SPRTN levels during ER stress, and increased sensitivity to ER stress in SPRTN-depleted cells. These findings suggest an essential role for SPRTN in the ER stress response and provide new insights into HCC pathogenesis. This newly discovered function of SPRTN could significantly enhance our understanding and treatment of HCC.

Mechanism of cis-Nerolidol-Induced Bladder Carcinoma Cell Death.

Nerolidol is a naturally occurring sesquiterpene alcohol with multiple properties, including antioxidant, antibacterial, and antiparasitic activities. A few studies investigating the antitumor properties of nerolidol have shown positive results in both cell culture and mouse models. In this study, we investigated the antitumor mechanism of cis-nerolidol in bladder carcinoma cell lines. The results of our experiments on two bladder carcinoma cell lines revealed that nerolidol inhibited cell proliferation and induced two distinct cell death pathways. We confirmed that cis-nerolidol induces DNA damage and ER stress. A mechanistic study identified a common cAMP, Ca2+, and MAPK axis involved in signal propagation and amplification, leading to ER stress. Inhibition of any part of this signaling cascade prevented both cell death pathways. The two cell death mechanisms can be distinguished by the involvement of caspases. The early occurring cell death pathway is characterized by membrane blebbing and cell swelling followed by membrane rupture, which can be prevented by the inhibition of caspase activation. In the late cell death pathway, which was found to be caspase-independent, cytoplasmic vacuolization and changes in cell shape were observed. cis-Nerolidol shows promising antitumor activity through an unorthodox mechanism of action that could help target resistant forms of malignancies, such as bladder cancer.

Apoptotic function of human PMS2 compromised by the nonsynonymous single-nucleotide polymorphic variant R20Q.

Mismatch repair (MMR) corrects replication errors during DNA synthesis. The mammalian MMR proteins also activate cell cycle checkpoints and apoptosis in response to persistent DNA damage. MMR-deficient cells are resistant to cisplatin, a DNA cross-linking agent used in chemotherapy, because of impaired activation of apoptotic pathways. It is shown that postmeiotic segregation 2 (PMS2), an MMR protein, is required for cisplatin-induced activation of p73, a member of the p53 family of transcription factors with proapoptotic activity. The human PMS2 is highly polymorphic, with at least 12 known nonsynonymous codon changes identified. We show here that the PMS2(R20Q) variant is defective in activating p73-dependent apoptotic response to cisplatin. When expressed in Pms2-deficient mouse fibroblasts, human PMS2(R20Q) but not PMS2 interfered with the apoptotic response to cisplatin. Correspondingly, PMS2 but not PMS2(R20Q) enhanced the cytotoxic effect of cisplatin measured by clonogenic survival. Because PMS2(R20Q) lacks proapoptotic activity, this polymorphic allele may modulate tumor responses to cisplatin among cancer patients.

Blood pressure dipping and salivary cortisol as markers of fatigue and sleep deprivation in staff anesthesiologists.

Anesthesiologists often work extended duty shifts that result in acute and chronic sleep loss and circadian disruption. Stress caused by sleep deprivation, together with excessive workload could contribute to acute increases in blood pressure (BP) and sympathetic nervous system activity. Non-dipping pattern of BP is considered an additional risk factor for cardiovascular events and target organ damage. We hypothesized that there would be significant changes of cardiovascular parameters when comparing work on call during the 24-hour in-hospital shift (24-HD) versus ordinary working day (8-HD) combined with changes of dipping pattern and altered diurnal cortisol secretion, measured by salivary cortisol (SC). Following local Medical Ethics Committee approval, 12 out of 36 staff anesthesiologists (8 male, 4 female), 33-61 years old, participated in this study. Ambulatory BP monitor was used for noninvasive 24-hour ambulatory BP and heart rate (HR) monitoring. Each participant was monitored continuously during the 8-HD, as well as during the 24-HD. Saliva for analysis of cortisol levels was collected six times a day (at 8 am, 11 am, 2 pm, 5pm, 8pm, and 11 pm) both during 8-HD and on 24-HD. There was a significant decrease in number of diastolic dippers on call vs. diastolic dippers on ordinary working day (4/12 vs. 10/12, p=0.036), and non significant decrease of systolic dippers (3/12 vs. 7/12, p =0.214). There were no significant differences in SC values between 8-HD and 24-HD at all observed time points. However, the SC values measured during the night were markedly elevated on both days compared with reference values and the shapes of SC curves were altered. The lack of diastolic BP dipping could be more sensitive indicator of stress among staff anesthesiologists than systolic BP dipping. The shape of SC diurnal curve in terms of elevated night values could be another indicator of their chronic fatigue.

Postexercise hypotension in moderately trained athletes after maximal exercise.

PURPOSE: A similar postexercise hypotension (PEH) has been reported in sedentary and mildly endurance-trained individuals of both sexes after a single dynamic submaximal exercise. In endurance-trained men, the hypotension was associated with a reduction of cardiac output, whereas the peripheral vasodilatation was the main mechanism of this fall in other groups. The present study investigated the occurrence and mechanisms of PEH after a short maximal exercise in professional soccer players with greater endurance capacity than previously reported in PEH studies. METHODS: Arterial blood pressure, cardiac output (Q), heart rate (HR), and diffusing lung capacity for carbon monoxide (DLCO) before and 30 and 60 min after short maximal field exercise were studied in 20 professional soccer players. RESULTS: Diastolic blood pressure (DBP) and systolic blood pressure (SBP), Q, stroke volume, and DLCO decreased, whereas HR increased at both times after exercise. Decreases in DBP were greater in subjects with lesser VO2max (r = -0.73, P = 0.0001), whereas SBP was more decreased the higher it was at baseline (r = 0.51, P = 0.023). Total peripheral resistance (TPR) did not change significantly after exercise. CONCLUSION: These findings indicate that, in moderately trained athletes, postexercise hypotension is associated primarily with reduced cardiac output because of reduced stroke volume, suggesting venous pooling. In addition, the occurrence of hypotension is more frequent in trained subjects with lower cardiopulmonary fitness level or higher resting SBP.

SPRTN is a mammalian DNA-binding metalloprotease that resolves DNA-protein crosslinks.

Ruijs-Aalfs syndrome is a segmental progeroid syndrome resulting from mutations in the SPRTN gene. Cells derived from patients with SPRTN mutations elicit genomic instability and people afflicted with this syndrome developed hepatocellular carcinoma. Here we describe the molecular mechanism by which SPRTN contributes to genome stability and normal cellular homeostasis. We show that SPRTN is a DNA-dependent mammalian protease required for resolving cytotoxic DNA-protein crosslinks (DPCs)- a function that had only been attributed to the metalloprotease Wss1 in budding yeast. We provide genetic evidence that SPRTN and Wss1 function distinctly in vivo to resolve DPCs. Upon DNA and ubiquitin binding, SPRTN can elicit proteolytic activity; cleaving DPC substrates and itself. SPRTN null cells or cells derived from patients with Ruijs-Aalfs syndrome are impaired in the resolution of covalent DPCs in vivo. Collectively, SPRTN is a mammalian protease required for resolving DNA-protein crosslinks in vivo whose function is compromised in Ruijs-Aalfs syndrome patients.

The impact of IL-6 and IL-28B gene polymorphisms on treatment outcome of chronic hepatitis C infection among intravenous drug users in Croatia.

BACKGROUND: Several genes and their single nucleotide polymorphisms (SNPs) are associated with either spontaneous resolution of hepatitis C infection or better treatment-induced viral clearance. We tested a cohort of intravenous drug users (IVDU) diagnosed with chronic hepatitis C virus (HCV) for treatment response and its association with the SNPs in the interleukin-6 (rs1800795-IL6) and the interleukin-28B (rs12979860-IL28B) genes. METHODS: The study included 110 Croatian IVDU positive for anti-HCV antibody. Genotyping was performed by polymerase chain reaction (PCR) based approach. Patients were treated by standard pegylated-interferon/ribavirin and followed throughout a period of four years, during which sustained virological response (SVR) was determined. All data were analysed with statistical package SPSS 19.0 (IBM Corp, Armonk, NY, USA) and PLINK v1.07 software. RESULTS: Patients showed a significantly better response to treatment according to the number of copies of the C allele carried at rs1800795-IL6 (P = 0.034). All but one of the patients with CC genotype achieved SVR (93%), whereas the response rate of patients with GG genotype was 64%. The association of rs1800795-IL6 with SVR status remained significant after further adjustment for patients' age, fibrosis staging, and viral genotype (OR 2.15, 95% CI 1.16-4.68, P = 0.019). Distributions of allele frequencies at the locus rs12979860-IL28B among the study cohort and the underlying general population were suggestive of a protective effect of CC genotype in acquiring chronic hepatitis C in the Croatian IVDU population. DISCUSSION: The rs1800795-IL6 polymorphism is associated with positive response to treatment in IVDU patients positive for HCV infection. A protective role of rs12979860-IL28B CC genotype in acquiring chronic hepatitis C is suggested for Croatian IVDU population.

Spleen volume and blood flow response to repeated breath-hold apneas.

The purpose of this study was 1) to answer whether the reduction in spleen size in breath-hold apnea is an active contraction or a passive collapse secondary to reduced splenic arterial blood flow and 2) to monitor the spleen response to repeated breath-hold apneas. Ten trained apnea divers and 10 intact and 7 splenectomized untrained persons repeated five maximal apneas (A1-A5) with face immersion in cold water, with 2 min interposed between successive attempts. Ultrasonic monitoring of the spleen and noninvasive cardiopulmonary measurements were performed before, between apneas, and at times 0, 10, 20, 40, and 60 min after the last apnea. Blood flows in splenic artery and splenic vein were not significantly affected by breath-hold apnea. The duration of apneas peaked after A3 (143, 127, and 74 s in apnea divers, intact, and splenectomized persons, respectively). A rapid decrease in spleen volume ( approximately 20% in both apnea divers and intact persons) was mainly completed throughout the first apnea. The spleen did not recover in size between apneas and only partly recovered 60 min after A5. The well-known physiological responses to apnea diving, i.e., bradycardia and increased blood pressure, were observed in A1 and remained unchanged throughout the following apneas. These results show rapid, probably active contraction of the spleen in response to breath-hold apnea in humans. Rapid spleen contraction and its slow recovery may contribute to prolongation of successive, briefly repeated apnea attempts.

Carpal tunnel syndrome is associated with high fibrinogen and fibrinogen deposits.

BACKGROUND: Idiopathic carpal tunnel syndrome (ICTS) is a common entrapment neuropathy. Some cases of ICTS are linked to mutations of the transthyretin gene, whereas others are associated with systemic amyloidosis. The majority of ICTS cases are of unknown etiology. OBJECTIVE: To study molecular mechanisms of ICTS development. METHODS: A total of 71 ICTS patients and 68 control subjects were included in the study. The fibrinogen level was determined before surgery and its deposition in the transversal carpal ligament (TCL) was detected by immunohistochemistry, Western blot, and mass spectrometry. Fibrinogen interaction with other proteins was studied by immunoprecipitation assay. RESULTS: Plasma levels of the proinflammatory and hemostatic protein fibrinogen are elevated in ICTS patients. Other measured systemic inflammatory markers were not affected, and local inflammatory responses in TCL were absent. ICTS patients have shorter bleeding times, probably because of the elevated plasma levels of fibrinogen. Polymorphisms of the fibrinogen B promoter region were previously associated with increased plasma fibrinogen, but this association was not observed among patients with ICTS. Interestingly, we detected fibrinogen deposits in the TCL, whereas transcriptional activity of the fibrinogen genes was low. Amyloidogenic proteins, including transthyretin and α-synuclein, were also found in the TCL, whereas their local transcriptional activity was rather high. Finally, we demonstrated that fibrinogen interacts with transthyretin and α-synuclein in TCL lysates. CONCLUSION: Our data indicate that fibrinogen and other aggregation-prone proteins have potentially important roles in the pathogenesis of ICTS.

Mutations in SPRTN cause early onset hepatocellular carcinoma, genomic instability and progeroid features.

Age-related degenerative and malignant diseases represent major challenges for health care systems. Elucidation of the molecular mechanisms underlying carcinogenesis and age-associated pathologies is thus of growing biomedical relevance. We identified biallelic germline mutations in SPRTN (also called C1orf124 or DVC1) in three patients from two unrelated families. All three patients are affected by a new segmental progeroid syndrome characterized by genomic instability and susceptibility toward early onset hepatocellular carcinoma. SPRTN was recently proposed to have a function in translesional DNA synthesis and the prevention of mutagenesis. Our in vivo and in vitro characterization of identified mutations has uncovered an essential role for SPRTN in the prevention of DNA replication stress during general DNA replication and in replication-related G2/M-checkpoint regulation. In addition to demonstrating the pathogenicity of identified SPRTN mutations, our findings provide a molecular explanation of how SPRTN dysfunction causes accelerated aging and susceptibility toward carcinoma.

No association between histamine N-methyltransferase functional polymorphism Thr105Ile and Alzheimer's disease.

Several abnormalities, including lower histamine levels in brain, elevated serum histamine and degeneration of histaminergic neurons in tuberomammillary nucleus, were described in the histaminergic system of patients with Alzheimer's disease (AD). Histamine is a central neurotransmitter with several functions in brain including regulation of memory, cognition, locomotion, and is degraded in part by histamine N-methyltransferase (HNMT). A common Thr105Ile polymorphism within HNMT gene results in decreased enzyme activity. The Thr105Ile polymorphism was associated with Parkinson's disease, essential tremor, attention-deficit hyperactivity disorder (ADHD), asthma and alcoholism, thus we tested possible association of HNMT functional polymorphism with AD. We have tested 256 AD cases and 1190 healthy controls of Croatian origin. Thr105Ile polymorphism was determined by TaqMan RT-PCR Genotyping Assay and EcoRV digestion. Prevalence of functional HNMT polymorphism among all tested groups was similar and frequency of less active Ile105 variant was 11.5% among AD patients and 13.4% for healthy controls (p=0.26, X(2)=1.25). Our results indicate lack of the association of HNMT Thr105Ile functional polymorphism with Alzheimer's disease.

Slower cortisol response during ACTH stimulation test in autistic children.

Autism is a hereditary, pervasive neurodevelopmental disorder that starts early in life. The main characteristics of the autism are impairment in social interactions, difficulties in adapting to novel environmental situations and improper reaction to stress. Since the Hypothalamic-Pituitary-Adrenocortical (HPA) axis plays a key role in the response to stress and because the previous research found abnormalities in HPA system, we conducted a study to test several elements of the HPA axis. Because autism is a heritable disorder, autistic subjects were studied as well as their parents. Cortisol circadian rhythm, cortisol daily secretion and its suppression response to dexamethasone had been measured from saliva or urine samples of the autistic children and their parents. Cortisol secretion response after ACTH stimulation was done with the autistic children only. The cortisol elevation after ACTH stimulation among the autistic individuals was slower (P = 0.017) than in healthy controls. No differences were found in salivary cortisol circadian rhythm or suppression response, as well as in cortisol daily excretion. These data indicate that, compared to healthy subjects, autistic individuals have fine differences in cortisol response to ACTH stimulation or possibly to other types of stress.

Aerobic exercise before diving reduces venous gas bubble formation in humans.

We have previously shown in a rat model that a single bout of high-intensity aerobic exercise 20 h before a simulated dive reduces bubble formation and after the dive protects from lethal decompression sickness. The present study investigated the importance of these findings in man. Twelve healthy male divers were compressed in a hyperbaric chamber to 280 kPa at a rate of 100 kPa min(-1) breathing air and remaining at pressure for 80 min. The ascent rate was 9 m min(-1) with a 7 min stop at 130 kPa. Each diver underwent two randomly assigned simulated dives, with or without preceding exercise. A single interval exercise performed 24h before the dive consisted of treadmill running at 90% of maximum heart rate for 3 min, followed by exercise at 50% of maximum heart rate for 2 min; this was repeated eight times for a total exercise period of 40 min. Venous gas bubbles were monitored with an ultrasonic scanner every 20 min for 80 min after reaching surface pressure. The study demonstrated that a single bout of strenuous exercise 24h before a dive to 18 m of seawater significantly reduced the average number of bubbles in the pulmonary artery from 0.98 to 0.22 bubbles cm(-2)(P= 0.006) compared to dives without preceding exercise. The maximum bubble grade was decreased from 3 to 1.5 (P= 0.002) by pre-dive exercise, thereby increasing safety. This is the first report to indicate that pre-dive exercise may form the basis for a new way of preventing serious decompression sickness.