Enzymatic Analysis of Recombinant Japanese Encephalitis Virus NS2B(H)-NS3pro Protease with Fluorogenic Model Peptide Substrates

Background Japanese encephalitis virus (JEV), a member of the Flaviviridae family, causes around 68,000 encephalitis cases annually, of which 20–30% are fatal, while 30–50% of the recovered cases develop severe neurological sequelae. Specific antivirals for JEV would be of great importance, particularly in those cases where the infection has become persistent. Being indispensable for flaviviral replication, the NS2B-NS3 protease is a promising target for design of anti-flaviviral inhibitors. Contrary to related flaviviral proteases, the JEV NS2B-NS3 protease is structurally and mechanistically much less characterized. Here we aimed at establishing a straightforward procedure for cloning, expression, purification and biochemical characterization of JEV NS2B(H)-NS3pro protease. Methodology/Principal Findings The full-length sequence of JEV NS2B-NS3 genotype III strain JaOArS 982 was obtained as a synthetic gene. The sequence of NS2B(H)-NS3pro was generated by splicing by overlap extension PCR (SOE-PCR) and cloned into the pTrcHisA vector. Hexahistidine-tagged NS2B(H)-NS3pro, expressed in E. coli as soluble protein, was purified to >95% purity by a single-step immobilized metal affinity chromatography. SDS-PAGE and immunoblotting of the purified enzyme demonstrated NS2B(H)-NS3pro precursor and its autocleavage products, NS3pro and NS2B(H), as 36, 21, and 10 kDa bands, respectively. Kinetic parameters, Km and kcat, for fluorogenic protease model substrates, Boc-GRR-amc, Boc-LRR-amc, Ac-nKRR-amc, Bz-nKRR-amc, Pyr-RTKR-amc and Abz-(R)4SAG-nY-amide, were obtained using inner filter effect correction. The highest catalytic efficiency kcat/Km was found for Pyr-RTKR-amc (kcat/Km: 1962.96±85.0 M−1 s−1) and the lowest for Boc-LRR-amc (kcat/Km: 3.74±0.3 M−1 s−1). JEV NS3pro is inhibited by aprotinin but to a lesser extent than DEN and WNV NS3pro. Conclusions/Significance A simplified procedure for the cloning, overexpression and purification of the NS2B(H)-NS3pro was established which is generally applicable to other flaviviral proteases. Kinetic parameters obtained for a number of model substrates and inhibitors, are useful for the characterization of substrate specificity and eventually for the design of high-throughput assays aimed at antiviral inhibitor discovery

Current Research into Applications of Tomography for Fusion Diagnostics

Retrieving spatial distribution of plasma emissivity from line integrated measurements on tokamaks presents a challenging task due to ill-posedness of the tomography problem and limited number of the lines of sight. Modern methods of plasma tomography therefore implement a-priori information as well as constraints, in particular some form of penalisation of complexity. In this contribution, the current tomography methods under development (Tikhonov regularisation, Bayesian methods and neural networks) are briefly explained taking into account their potential for integration into the fusion reactor diagnostics. In particular, current development of the Minimum Fisher Regularisation method is exemplified with respect to real-time reconstruction capability, combination with spectral unfolding and other prospective tasks

Hidradenitis suppurativa : With special reference to carbon dioxide laser surgery

Hidradenitis suppurativa (HS) is a chronic inflammatory disease with recurrent abscesses. In most cases, it involves the axillar and anogenital regions. In industrialised countries, the prevalence is of 0.3% to 4%, and the disease is over-represented in young adult females. Various medical treatments have been used but they are seldom effective. Surgical treatment is recommended as soon as the condition is diagnosed, but wide excisions very well outside the clinical borders of activity, are mandatory. A simple local incision is of no value. According to Hurley's clinical classification, stage I consists of one or more abscesses with no sinus tract and cicatrisation and stage II consists of one or more widely separated recurrent abscesses, with a tract and cicatrisation. The severest cases (stage III) have multiple interconnected tracts and abscesses throughout an entire area. In this thesis we report a method in which horisontal vaporisation with carbon dioxide laser is used to remove the inflamed infiltrating abscesses with precision. Patients classified as Hurley stage H were selected consecutively, in 1989 and by the year 2000 that had undergone this treatment. Most patients were females, in their twenties and thirties and had had the disease for a mean of more than ten years. By using a radical but selective, tissue-sparing technique, we removed the inflamed, foreign body-like tissues of HS, including its squamous epitheliumlined and keratin- containing sinuses. Initially, we used a free hand and more surgeondependent technique that was later abandoned for a scanner assisted technique providing better accuracy, safety and faster ablation. The use of these carbon dioxide laser, rapid beam, optomechanical scanner systems in a continuous mode gives a fast and even ablation with better visualisation of the macro- pathology during surgery. The surgical results were satisfactory cosmetically, functionally and as regards quality of life. We believe that this technique offers a safe and efficient strategy for many colleagues who treat HS. Squamous cell carcinoma is a rare but serious complication of HS. To investigate this association and the risk of other malignancies, we performed a population-based retrospective cohort study on 2119 HS patients selected from a computerised database of hospital discharge diagnoses in Sweden during 1965-1997. We found a significantly increased risk of non- melanoma skin cancer in patients with HS. The risks of buccal cancer and primary liver cancer were also higher. Aerobic and anaerobic cultures from superficial and deep levels were taken during laser surgery in 24 patients. In all cases, bacterial cultures were positive for one or more specimens from at least one level and from deep levels in all but three cases. Sixteen species or subspecies were found. Staphylococcus OBS aureus OBS and coagulase-negative OBS staphylococci OBS (CNS) were the most frequently found species which suggests that CNS is a true pathogen. In our comparison of forty-two unrelated Swedish patients with HS and 250 controls we found no association with HLA-A, -B or -DRB1 alleles using the genetic tissue typing technique. Genetic factors associated with the HLA class I or II regions do not seem to contribute significantly to the possible genetic susceptibility of HS. We also compared the release of oxygen radicals and of primary granula from in vitro activated peripheral neutrophils from HS patients with inactive disease to that from a group of healthy controls. Our findings suggest that dysfunctional neutrophils may be involved in the pathogenesis of hidradenitis suppurativa

Shift in Occupational Risk for Basal Cell Carcinoma from Outdoor to Indoor Workers: A Large Population-based Case-control Register Study from Sweden

Basal cell carcinoma (BCC) is the most common form of cancer worldwide. Exposure of the skin to ultraviolet (UV) radiation, from sunlight and other sources, is the most important risk factor. The aim of this large-scale case-control study was to determine which occupations are associated with increased risk of BCC in Sweden. The case cohort comprised 74,247 patients with BCC and the control cohort comprised 574,055 subjects linked to population-based registers. Compared with the occupational category of farmers, foresters and gardeners we observed elevated risks of BCC for almost all occupational categories studied. Legal workers with odds ratio (OR) 2.69 (95% confidence interval (CI) 2.36–3.06), dentists OR 2.69 (95% CI 2.35–3.08) and physicians OR 2.47 (95% CI 2.24–2.74) had the highest risk for both sexes taken together. In conclusion, there appears to have been a change in the risk of BCC from outdoor to indoor occupations in Sweden, possibly related to exposure to UV radiation during leisure activities exceeding occupational sun exposure as the main cause of BCC in Sweden

Familial features affecting the melanoma risk in <i>CDKN2A</i>-negative melanoma families: a study based on the Swedish Cancer Registry

Familial features affecting the melanoma risk in CDKN2A-negative melanoma families: a study based on the Swedish Cancer Registr

Trend Shifts in Age-Specific Incidence for In Situ and Invasive Cutaneous Melanoma in Sweden

Simple Summary The incidence of invasive cutaneous melanoma (CM) is increasing in Sweden. The aim was to present age- and sex-specific trends of the age-standardised incidence and the average annual percentage change (AAPC) for in situ and invasive CM by analysing data obtained from the Swedish Melanoma Register and the Swedish Cancer registry for 35,350 in situ tumours and 59,932 CM. Trend shifts in age-specific incidence for in situ and invasive CM reflect a rise among both sexes since the 2000s and could be a result of more effective secondary prevention efforts and a higher awareness of CM. Background: The incidence of invasive cutaneous melanoma (CM) is increasing in Sweden. The aim was to present age- and sex-specific trends of the age-standardised incidence and the average annual percentage change (AAPC) for in situ and invasive CM. Methods: Joinpoint regression models were used to analyse data from the Swedish Cancer Register and the Swedish Melanoma Registry 1997-2018 (N = 35,350 in situ CM; 59,932 CM). Results: The AAPC of CM for women was 4.5 (4.1-5.0; p &amp;lt; 0.001) for the period 1997-2018. For men, the APCC was 4.2 (3.0-5.4; p &amp;lt; 0.001), with a significantly higher annual percentage change (APC) for the period 2000-2018 (5.0; 4.6-5.4; p &amp;lt; 0.001) compared to 1997-1999. An increasing annual incidence of CM &amp;lt;= 0.6 mm and 0.7 mm Breslow tumour thickness was found for men with a significant incidence shift for the period 2006-2015, respectively. Similarly for women, with a significantly higher APC for CM &amp;lt;= 0.6 mm from 2005. The incidence of intermediate thick CM (2.1-4.0 mm) has not increased since 2011. The incidence of CM &amp;gt; 4.0 mm has been increasing among both sexes, with a significantly lower APC among women from 2005. Conclusions: The incidence of in situ and low-risk CM &amp;lt;= 1.0 mm in tumour thickness has been rising among both sexes since the 2000s.Funding Agencies|Swedish Cancer SocietySwedish Cancer Society [190278Fk01H, 210406]; Cancer Research Foundations of Radiumhemmet [181083]; Swedish Medical Society [SLS-934511, SLS-961466]; Swedish Society for Medical Research; KI Funds [FS-2020:0007]; Hudfonden [4-1880/2020]; Sigurd and Elsa Golje Memorial Fund [LA-2020-0148]; Folkfonden; Castenback Fund; S.R. Gorthon foundation; Krapperup foundation [0029-2020]; FOU Region Skane [815731]; ALF Lund University</p

MiR-130a Acts as a Tumor Suppressor MicroRNA in Cutaneous Squamous Cell Carcinoma and Regulates the Activity of the BMP/SMAD Pathway by Suppressing ACVR1

Cutaneous Squamous Cell Carcinoma (cSCC) is a malignant neoplasm of the skin resulting from the accumulation of somatic mutations due to solar radiation. It is one of the fastest increasing malignancies and it represents a particular problem among immunosuppressed individuals. MicroRNAs (miRNAs) are short non-coding RNAs that regulate the expression of protein-coding genes at the posttranscriptional level. Here we identify miR-130a to be downregulated in cSCC compared with healthy skin and with precancerous lesions (actinic keratosis) and demonstrate that it is regulated at the transcriptional level by HRAS and MAPK-signaling. We report that miR-130a suppresses the growth of cSCC xenografts in mice. We demonstrate that overexpression of miR-130a suppresses long-term capacity of growth, cell motility and invasion ability in human cSCC cell lines. Mechanistically, miR-130a directly targets Activin A receptor, type I (ACVR1/ALK2) and changes in miR-130a levels result in the diminished activity of BMP/SMAD1 pathway via ACVR1. These data reveal a link between activated MAPK-signaling and decreased expression of miR-130a, which acts as a tumor suppressor miRNA in cSCC and contributes to a better understanding of molecular processes in malignant transformation of epidermal keratinocytes

Increasing melanoma incidence and survival trend shifts with improved melanoma-specific survival between 1990 and 2020 in Sweden

BACKGROUND: The melanoma-specific survival (MSS) is heterogenous between stages and is highly dependent on the T stage for primary localized disease. New systemic therapies for metastatic cutaneous melanoma (CM) have been introduced since 2012 in Sweden.OBJECTIVES: To analyse the incidence and MSS time trends between 1990 and 2020 in Sweden.METHODS: Nationwide, population-based, and prospectively collected clinico-pathological data on invasive CM from the Swedish Melanoma Registry (SweMR) were analysed for survival trends between 1990 and 2020 by using Kaplan-Meier curves and Cox Proportional Hazard ratios.RESULTS: In total, 77 036 primary invasive CM were diagnosed in 70 511 patients in Sweden between 1990 and 2020. The 5-year MSS was 88.9% (88.3-89.4) 1990-2000, 89.2% 2001-2010, and 93.0% (92.7-93.9) 2011-2020. The odds ratios for being diagnosed with nodular melanomas (vs. superficial spreading melanoma) was significantly reduced by 20% (2001-2010) and by 46% (2011-2020 vs. 1990-2000). Overall, the MSS improved over both diagnostic periods (2001-2010 and 2011-2020) vs the reference period 1990-2000 among men and women, respectively, (HRmen, 2001-2010: 0.89, 95% CI 0.82-0.96; HRmen, 2011-2020: 0.62, 95% CI 0.56-0.67. HRwomen, 2001-2010: 0.82, 95% CI 0.74-0.91; HRwomen, 2011-2020: 0.62, 95% CI 0.56-0.70). The risk of CM-death was significantly lower in all age groups for both men and women in the most recent diagnostic period (2011-2020 vs. 1990-2000).CONCLUSIONS: The results are emphasizing the improved MSS among both men and women in Sweden. The MSS improvements, specifically for the period 2011-2020, may be correlated to the introduction of new systemic therapies and are here shown for the first time in detail for Sweden

Multiple primary melanoma incidence trends over five decades, a nation-wide population-based study

BACKGROUND: Over the past decades many regions have experienced a steady increase in the incidence of cutaneous melanoma. Here, we report on incidence trends for subsequent primary melanoma.METHODS: In this nationwide population-based study, patients diagnosed with a first primary cutaneous melanoma reported to the Swedish Cancer Registry, were followed for up to ten years for a diagnosis of subsequent primary melanoma. Patients were grouped with patients diagnosed with first melanoma in the same decade (1960s, 1970s, 1980s, 1990s and 2000s, respectively). Frequencies, incidence rates (IRs), standardized incidence ratios (SIRs) and 95% confidence intervals (CIs) for second melanomas were calculated. All tests of statistical significance were two-sided.RESULTS: 54,884 patients with melanoma were included and 2,469 were diagnosed, within ten years, with subsequent melanomas. Over the five decades there was a statistically significant steady increase in the frequency, IR and SIR for second primary melanoma. For example, in the 1960s cohort, 2 melanomas increased statistically significantly and was 0.0% in the 1960s and rose to 18.0% in the 2000s (P <.001).CONCLUSIONS: This is the first study to evaluate and report on a rising trend for subsequent primary melanoma. Additional primary melanomas worsen the patients' survival and precautions are needed to turn this steep upgoing trend

CDKN2A genetic testing in melanoma-prone families in Sweden in the years 2015–2020 : implications for novel national recommendations

Background: Inherited pathogenic variants (PVs) in the CDKN2A gene are among the strongest known risk factors for cutaneous melanoma. Carriers are at high risks to develop multiple primary melanomas and other cancers, in particular pancreatic cancer. In this study, the CDKN2A testing, carried out in Sweden in the years 2015–2020, was evaluated. Materials and methods: Included families had (1) three or more cases of melanoma and/or pancreatic cancer, (2) two melanomas in first-degree relatives, the youngest case &lt;55 years or (3) individuals with three or more multiple primary melanomas, the first before the age of 55 years, and no other affected family members. The included families had at least one affected member that had been tested for CDKN2A PVs. Results: In total, 403 families were included, whereof 913 family members had been diagnosed with cutaneous melanoma and 129 with pancreatic cancer, 33 (8.2%) were found to have PVs in CDKN2A. Frequencies ranged from 0.9% in families with only two melanomas to 43.2% in families with three or more melanoma cases and pancreatic cancer (p &lt; 0.001). The frequency of PVs ranged from 2.1% to 16.5% in families where the youngest case was ≥55 years or &lt;35 years (p = 0.040). In families with or without CDKN2A PVs, 37.6% and 10.0% had melanoma cases that had died from melanoma, respectively (p &lt; 0.001). Discussion: Significant differences were seen in the frequencies of CDKN2A PVs, dependent on numbers or age at diagnosis of melanomas and diagnoses of pancreatic cancers in the family. Further, melanoma cases belonging to families that tested positive for CDKN2A PVs had a significantly higher mortality. To summarize, the current evaluation shows that, with adequately selected criteria to guide genetic testing, CDKN2A PVs are identified at significant frequencies. Identification of carrier families is of importance to ensure that members are enrolled in a preventive surveillance program