Transcriptomic analysis of resistance and short-term induction response to pyrethroids, in Anopheles coluzzii legs.

BACKGROUND: Insecticide-treated bed nets and indoor residual spraying comprise the major control measures against Anopheles gambiae sl, the dominant vector in sub-Saharan Africa. The primary site of contact with insecticide is through the mosquitoes' legs, which represents the first barrier insecticides have to bypass to reach their neuronal targets. Proteomic changes and leg cuticle modifications have been associated with insecticide resistance that may reduce the rate of penetration of insecticides. Here, we performed a multiple transcriptomic analyses focusing on An. coluzzii legs. RESULTS: Firstly, leg-specific enrichment analysis identified 359 genes including the pyrethroid-binder SAP2 and 2 other chemosensory proteins, along with 4 ABCG transporters previously shown to be leg enriched. Enrichment of gene families included those involved in detecting chemical stimuli, including gustatory and ionotropic receptors and genes implicated in hydrocarbon-synthesis. Subsequently, we compared transcript expression in the legs of a highly resistant strain (VK7-HR) to both a strain with very similar genetic background which has reverted to susceptibility after several generations without insecticide pressure (VK7-LR) and a lab susceptible population (NG). Two hundred thirty-two differentially expressed genes (73 up-regulated and 159 down-regulated) were identified in the resistant strain when compared to the two susceptible counterparts, indicating an over-expression of phase I detoxification enzymes and cuticular proteins, with decrease in hormone-related metabolic processes in legs from the insecticide resistant population. Finally, we analysed the short-term effect of pyrethroid exposure on An. coluzzii legs, comparing legs of 1 h-deltamethrin-exposed An. coluzzii (VK7-IN) to those of unexposed mosquitoes (VK7-HR) and identified 348 up-regulated genes including those encoding for GPCRs, ABC transporters, odorant-binding proteins and members of the divergent salivary gland protein family. CONCLUSIONS: The data on An. coluzzii leg-specific transcriptome provides valuable insights into the first line of defense in pyrethroid resistant and short-term deltamethrin-exposed mosquitoes. Our results suggest that xenobiotic detoxification is likely occurring in legs, while the enrichment of sensory proteins, ABCG transporters and cuticular genes is also evident. Constitutive resistance is primarily associated with elevated levels of detoxification and cuticular genes, while short-term insecticide-induced tolerance is linked with overexpression of transporters, GPCRs and GPCR-related genes, sensory/binding and salivary gland proteins.

The evolution of mapping and ablation techniques in the treatment of atrial tachycardias occurring after atrial fibrillation ablation.

PURPOSE: A possible consequence of atrial fibrillation (AF) ablation is the occurrence of organized atrial tachycardias (ATs). ATs after AF ablation (ATAAF) may be more symptomatic than AF itself, thus necessitating catheter ablation. We evaluated the prognostic significance of clinical and invasive characteristics for long-term sinus rhythm (SR) maintenance following ATAAF ablation and assessed the effect of technological developments on these results. METHODS AND RESULTS: Fifty-six consecutive patients with symptomatic ATAAF participated in the study and 114 ATAAF were revealed (2.04 ± 0.93 per patient). Sixty-eight ATAAF (60%) were macroreentrant and 33 (29%) were microreentrant circuits, while 13 (11%) were focal automatic tachycardias. The mean follow-up was 40 ± 18 months with 34 (61%) patients maintaining SR. Treatment with contact force (CF) catheters and EnSite AutoMap module (n = 11) showed significantly better AT/AF free rates at 1-year follow-up (10/11, 91%) compared with treatment using CF catheters but not AutoMap module (n = 13) (8/13, 62%) and treatment with use of neither of these modalities (n = 32) (16/32, 50%). Among patients with macroreentrant circuits around the mitral annulus or left atrial roof (n = 38), the group treated with complete linear lesions in anatomical isthmuses (n = 25) showed significantly better SR maintenance (19/25, 76%) compared with patients (n = 13) treated by empirical ablation in critical functional areas (6/13, 46%). CONCLUSIONS: Technology advancement contributes substantially to long-term success in SR maintenance, by achieving detailed mapping and more effective ablation of ATAAF. The targeting of macroreentrant circuits by creating anatomical linear lesions appears to provide better results.

Efficient genome editing in the olive fruit fly, Bactrocera oleae.

The olive fruit fly, Bactrocera oleae, causes great damage to the quality and quantity of olive production worldwide. Pest management approaches have proved difficult for a variety of reasons, a fact that has brought about a need for alternative tools and approaches. Here we report for the first time in B. oleae the development of the clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) gene editing tool, using the well-known eye colour marker gene scarlet. Two synthetic guide RNAs targeting the coding region of the scarlet gene were synthesized and shown to work efficiently in vitro. These reagents were then microinjected along with purified Cas9 protein into early-stage embryos. Successful CRISPR-induced mutations of both copies of the scarlet gene showed a striking yellow eye phenotype, indicative of gene disruption. Multiple successful CRISPR events were confirmed by PCR and sequencing. The establishment of an efficient CRISPR-based gene editing tool in B. oleae will enable the study of critical molecular mechanisms in olive fruit fly biology and physiology, including the analysis of insecticide resistance mechanisms and the discovery of novel insecticide targets, as well as facilitate the development of novel biotechnology-based pest control strategies.

Vitamin D in mild cognitive impairment and Alzheimer's disease. A study in older Greek adults.

BACKGROUND: In recent years, accumulating evidence has linked vitamin D deficiency to cognitive dysfunction and dementia. This study aimed at determining the relevance of serum 25-hydroxyvitamin D concentrations in mild cognitive impairment (MCI) and Alzheimer's disease (AD) in older Greek adults. It also examined whether the vitamin D level could be considered a predisposing factor for conversion from MCI to AD. METHODS: The study enrolled 350 subjects aged 65 years and over, allocated into three groups consisting of 103 healthy subjects (HS), 109 individuals with MCI, and 138 patients with AD, respectively. Serum 25-hydroxyvitamin D [25(OH)D] concentrations, measured in ng/ml, were determined by electrochemiluminescence, and we used the Mini-Mental State Examination (MMSE) and the Cambridge Cognition Examination (CAMGOG) to evaluate the subjects' cognitive status. One follow-up examination was performed for the MCI patients 30 months ± three months after the initial evaluation. RESULTS: Compared to HS, serum 25(OH)D levels were significantly decreased in individuals with MCI (p =0.012) and patients with AD (p <0.001). Moreover, serum 25(OH)D concentrations were significantly decreased in patients with AD compared to individuals with MCI (p =0.003) and also significantly lower in individuals with MCI who progressed to AD compared to those who remained MCI (p =0.028). After adjusting for confounders, multivariate analysis revealed that an increase of vitamin D concentration by one ng/mL reduces the risk of MCI by 4 % (OR =0.96, 95 % CI =0.92-0.99, p =0.006), the risk of AD by 8 % (OR =0.92, 95 % CI =0.89-0.95, p <0.001), and in an individual with MCI reduces the risk of conversion to AD by 10 % (OR =0.90, 95 % CI =0.83-0.96, p =0.003). CONCLUSIONS: The present study reveals that serum vitamin D levels are significantly decreased in subjects with MCI and patients with AD compared to HS. Additionally, individuals with MCI who progressed to AD presented significantly lower vitamin D levels than those who remained MCI. These results suggest that preserving adequate vitamin D status in older adults could delay or prevent cognitive decline. HIPPOKRATIA 2020, 24(3): 120-126.

Long-term effects of prolonged-release fampridine in cognitive function, fatigue, mood and quality of life of MS patients: The IGNITE study.

BACKGROUND: Studies have reported conflicting results regarding the potential benefit of prolonged release (PR) fampridine in other domains besides walking. Moreover, only a small number of studies have explored long- term effects of PR fampridine. The aim of this study was to assess cognitive function, quality of life, mood and fatigue in MS patients treated with fampridine after 6 and 12 months of treatment. METHODS: IGNITE was an observational, open label study. Subjects were examined with the timed 25-ft walk (T25FW) and the BICAMS battery and were asked to complete the Multiple Sclerosis Impact Scale (MSIS-29), Modified Fatigue Impact Scale (MFIS), Beck Depression Inventory-II (BDI-II) and MS International Quality-of-Life questionnaire (MUSIQOL) at baseline and at weeks 24 and 48. Patients were sub-grouped into responders (n:40) and non-responders (n:20) according to T25FW performance after 2 weeks on treatment. RESULTS: After 6 months, statistically significant improvement was observed on T25FW (p < .001), SDMT (p < .001) and MSIS29 (p < .001), for responders. After 1 year on treatment, statistically significant improvement was observed in T25FW (p < .001), MSIS29 (p = .004), SDMT (p < .001) and MUSIQOL (p = .03) for responders. There were no statistically significant improvements for the non-responders. CONCLUSIONS: PR Fampridine may have a beneficial effect on information processing speed though not on memory. Study data provide some evidence that fampridine treatment may reduce the impact of MS on daily activities and improve quality of life but has no effect on subjective fatigue and mood.

Genetic diversity of mycobacterium tuberculosis in northern Greece.

The aim of the present study was to describe the genetic diversity of Mycobacterium tuberculosis (M.tuberculosis) strains circulating in the region of Northern Greece. A total of thirty-seven M. tuberculosis clinical isolates were analysed by the spoligotyping method. According to the results, six clusters comprising seventeen strains were detected, and the remaining twenty strains showed unique patterns. The M.tuberculosis families according to SITVITWEB were distributed as follows: Haarlem (H) (27.0%); T (24.3%); Beijing (13.5%); Latin-America and Mediterranean (LAM) (5.4%) and S (2.7%). The remaining isolates (27%) did not match any isolates within the database and they were characterized as orphans. Regarding GenoType MTBDRplus results, two strains (5.4%) were Multi-Drug-Resistant, four strains (10.8%), were isoniazid monoresistant, while the remaining thirty-one strains (83.8%) were susceptible. In conclusion, in the region of Macedonia-Thrace (Northern Greece), there was high phylogenetic diversity among M. tuberculosis isolates. Molecular tools used and data presented can have regional and national impact on tuberculosis control.

Validation of the Brief International Cognitive Assessment for Multiple Sclerosis (BICAMS) in Greek population with multiple sclerosis.

BACKGROUND: Cognitive impairment is experienced by about 50% of patients with Multiple Sclerosis (MS) worldwide and affects their employment, disease management and quality of life in general. The Brief International Cognitive assessment for MS (BICAMS) is a brief, practical and potentially universal battery for cognitive impairment in MS patients. It consists of three tests: the Symbol Digit Modalities Test (SDMT), the California Verbal Learning Test-2 (CVLT-2) and the Brief Visuospatial Memory Test-Revised (BVMT-R). OBJECTIVE: The objective of this study was to validate the BICAMS in Greek MS patients and controls. METHODS: Forty four MS patients and seventy nine healthy control (HC) participants were recruited and tested. They were group matched for age, education, gender and also premorbid cognitive reserve. All of them completed the three tests of the BICAMS battery. Instead of CVLT-2, the Greek validated form (Greek Verbal Learning Test, GVLT), was used. In addition, cognitive reserve was assessed using the Cognitive Reserve Index questionnaire (CRIq) standardized for the Greek population. RESULTS: Significant difference was found in the performance of the two groups in all tests (p<0.0001, p<0.02, p<0.009 for SDMT, GVLT and BVMT-R respectively). Test-retest reliability was good for all the tests. Based on the criterion of 1 or more tests below the 5th percentile of healthy controls performance, 47% of patients were found impaired. CONCLUSIONS: The study provides validation of BICAMS in Greek population and therefore facilitates the use of this battery in clinical practice and in future studies of MS patients in Greece.

Levetiracetam as alternative treatment in Jeavons syndrome.

We report on a 25 year old female patient who had a diagnosis of Jeavons syndrome since her childhood. Although valproate led to seizure freedom, she developed persistent reproductive endocrine disorders attributed to this drug. The withdrawal of valproate in parallel with an initiation of levetiracetam monotherapy resulted in a maintenance of clinical remission and a resolution of these abnormalities. This case together with relevant literature data supports the view that the use of levetiracetam might be of benefit for female patients with Jeavons syndrome.

Coexistence of multiple sclerosis and ankylosing spondylitis: report of two cases.

BACKGROUND: Multiple sclerosis (MS) only rarely coexists with ankylosing spondylitis (AS). The optimal management of these patients represents a major challenge. METHODS: In the present study, we report 2 cases of AS with definite MS comorbidity. One of the AS-MS cases had received anti-TNFα treatment, which was discontinued due to exacerbation of the MS. In addition, we discuss 3 more AS cases with neurological symptoms and atypical white matter demyelinating MRI lesions after anti-TNFα treatment. DISCUSSION: Given the fact that anti-TNFα drugs can potentially exacerbate a latent MS or induce atypical demyelination in the central nervous system, they should be discouraged or discontinued in relevant cases. The remaining effective therapeutic options for MS are either contradictory for AS (interferon-ß), have no definite data regarding their safety/efficacy in AS (glatiramer acetate, azathioprine, natalizumab, fingolimod), or their efficacy in MS-AS is associated with increased treatment risks (rituximab). Any of these proposed treatments may require active patient's informed consent.

Fatal case of extensively drug-resistant Mycobacterium tuberculosis Beijing genotype infection in an injecting drug user, Athens, Greece, 2012.

We present the first fatal case of extensively drug-resistant tuberculosis (XDR-TB) in an injecting drug user (IDU) in Athens, Greece, co-infected with human immunodeficiency virus and hepatitis C virus and discuss the implications for public health. Despite immediate initiation of treatment, the patient's condition gradually deteriorated and he died 16 days after hospital admission because of multiple organ failure. The contact tracing investigation revealed no further infections among the patient's contacts.

Impact of the Gen-Probe Amplified MTD® Test on tuberculosis diagnosis in children.

OBJECTIVE: To evaluate the performance of the Gen-Probe Amplified MTD® Test (AMTD) for childhood tuberculosis (TB) diagnosis compared to conventional culture. DESIGN: We retrospectively studied 121 childhood cases (73 males; median age 7 years, range 1-16). Pulmonary samples (104/152, 68%) included gastric aspirates (n = 53), induced sputum samples (n = 43), bronchial aspirates and bronchoalveolar lavage (n = 8). Extra-pulmonary samples (48/152, 32%) included lymph nodes (n = 34) and other sterile fluids (n = 14). Specimens were examined using acid-fast bacilli (AFB) microscopy, AMTD and bacterial culture using BACTEC™ MGIT™ 960 and Löwenstein-Jensen (LJ) media. RESULTS: A clinical diagnosis of TB was made in 50/121 (41%) children (43/50 pulmonary disease). AFB microscopy was positive in 6%; Mycobacterium tuberculosis was recovered by culture from 16/50 (32%) and AMTD was positive in 29/50 (58%). AMTD sensitivity, specificity, positive predictive value and negative predictive value compared to culture were respectively 100%, 85%, 50% and 100%. For pulmonary vs. extra-pulmonary disease, the performance of AMTD compared to culture was respectively 100%, 77%, 46% and 100% vs. 100%, 97.5%, 75% and 100%. CONCLUSIONS: Nucleic acid amplification tests are more sensitive and very specific methods for the rapid detection of M. tuberculosis. The AMTD technique increases TB detection in children compared to conventional culture.

Transient epileptic amnesia in a memory clinic setting: a report of three cases.

Transient epileptic amnesia (TEA) is a recently described, typically misdiagnosed at presentation, distinctive syndrome, characterized by recurrent, brief attacks of memory loss in middle-aged or elderly people, accompanied by significant interictal memory difficulties. We describe the clinical, neuroimaging, electrophysiological, and neuropsychological characteristics of three patients fulfilling the proposed criteria for TEA, initially referred to our memory clinic for evaluation and treatment of probable dementia. Neuropsychological performance on admission and 6 months after treatment demonstrated a broad range of performance on measures of executive function, lower than expected visuospatial perception, and poor autobiographical memory. TEA should be included in the differential diagnosis of dementia to avoid misdiagnosis and needless medication. It is a benign and treatable condition, yet the associated decline in autobiographical and remote memory despite antiepileptic therapy poses challenges for further research.

Adaptation of Addenbrooke's Cognitive Examination-Revised for the Greek population.

BACKGROUND/PURPOSE: Addenbrooke's Cognitive Examination-Revised [1] is a highly sensitive and specific bedside test for the diagnosis of dementia. The aim of this study was to validate a Greek version of this simple, bedside instrument and to investigate its psychometric properties. METHOD: We translated and administered the Greek version of the ACE-R to 95 individuals comprising two groups: a dementia group (n=35) and an age-matched and education-matched healthy control group (n=60). RESULTS: Our results suggest high internal consistency and very good sensitivity, specificity and negative predictive value, but only adequate positive predictive value for the ACE-R, with excellent diagnostic accuracy. In our sample, the ratio of verbal fluency plus language scores to orientation plus memory (VLOM) subscore failed to discriminate patients in the target dementia group. CONCLUSION: The present findings support the usefulness of the ACE-R as a brief dementia screening tool in clinical practice. Further neuropsychologic evaluation is required for the differential diagnosis of dementia.

Tuberculosis in Greece: bacteriologically confirmed cases and anti-tuberculosis drug resistance, 1995-2009.

The Greek National Reference Laboratory for Mycobacteria is a major source of tuberculosis (TB)-related data for Greece, where the TB burden and epidemiology still need to be better defined. We present data regarding newly diagnosed TB cases and resistance to anti-TB drugs during the last 15 years in Greece. Although the total number of newly detected TB cases has declined, cases among immigrants are increasing. Resistance to first-line anti-TB drugs is widely prevalent, although stable or declining. The implementation of an efficient and effective countrywide TB surveillance system in Greece is urgently needed.

The prevalence of oligoclonal bands in the CSF of patients with primary neurodegenerative dementia.

BACKGROUND: Recent guidelines from the United States and Europe on the diagnosis and management of dementia include advice that younger patients with dementia should undergo CSF examination, which frequently includes analysis for oligoclonal bands (OCB). The presence of CNS specific OCB has traditionally been considered suggestive of an inflammatory aetiology, although the interpretation of such a finding in the presence of a normal CSF white cell count and protein is more difficult. METHODS: We reviewed retrospectively the prevalence of OCB, determined using agarose isoelectric focusing, in a series of 131 well characterised patients with a final diagnosis of a degenerative dementia who had undergone CSF examination. RESULTS: The mean age of the patients was 60.0 (SD 8.4) years. Seventy (53%) patients had Alzheimer's disease (AD), forty seven (36 %) had frontotemporal lobar degeneration (FTLD), seven (5%) had Dementia with Lewy bodies and the remaining seven (5%) patients had other rarer neurodegenerative dementias. Neuropathological examination had been performed in fifteen (11%) patients. CNS specific OCB were present in nine (7%) patients in this cohort, all of whom had normal CSF white cell counts: four AD patients (a prevalence of 6%), four FTLD patients (a prevalence of 9%), and one patient with Creutzfeldt-Jakob disease (a prevalence of 25%). Investigation of these patients, including two with neuropathologically verified AD and one with post-mortem confirmed CJD, did not reveal an alternative aetiology for their dementia. CONCLUSION: A central immune response can occur in primary neurodegenerative dementias, albeit uncommonly.

CRD-BP: a c-Myc mRNA stabilizing protein with an oncofetal pattern of expression.

The Coding Region Determinant-Binding Protein (CRD-BP) is an RRM and KH-domain-containing protein that recognizes specifically at least three RNAs. It binds to one of the two c-myc mRNA instability elements, to the 5'Un Translated Region (UTR) of the leader 3 IGF-II mRNA and to the oncofetal H19 RNA. CRD-BP has been assigned a role in stabilizing c-myc mRNA by preventing its endonucleolytic cleavage and in repressing the translation of the leader 3 IGF-II mRNA, the major embryonic species of this message. CRD-BP is normally expressed only in fetal tissues. However, its expression is detected in primary tumors and transformed cell lines of different origins. The vast majority of colon (80%) and breast (60%) tumors and sarcomas (73%) express CRD-BP whereas in other tumor types, for example prostate carcinomas, its expression is rare. CRD-BP expression has also been detected in benign tumors such as breast fibroadenomas, meningiomas and other benign mesenchymal tumors, implying a role for this gene in abnormal cell proliferation. In breast carcinomas, CRD-BP expression and or gene copy number gains in the region encompassing the c-myc locus were detected in approximately 75% of tumors, implying that the deregulated expression of c-myc may be more widespread than previously believed. Infiltrated lymph nodes, corresponding to CRD-BP-positive primary tumors, were also found positive indicating that monitoring for CRD-BP could prove useful for the detection and monitoring of disseminated disease.

C-MYC and IGF-II mRNA-binding protein (CRD-BP/IMP-1) in benign and malignant mesenchymal tumors.

Mouse coding region determinant-binding (mCRD-BP) and human IGF-II mRNA-binding 1 (hIMP-1) proteins are orthologous mRNA-binding proteins that recognize c-myc and IGF-II mRNA, respectively, and regulate their expression posttranscriptionally. Here, we confirm that human CRD-BP/IMP-1 binds to c-myc mRNA and that it is predominantly expressed in fetal tissues. Moreover, hCRD-BP/IMP-1 expression was detected in cell lines of neoplastic origin and in selected primary tumors. In a series of 33 malignant and 10 benign mesenchymal tumors, 73% and 40%, respectively, were found to express hCRD-BP/IMP-1. In particular, expression was significant in 14 Ewing's sarcomas, all of which were positive. The data suggest that hCRD-BP/IMP-1 plays a role in abnormal cell proliferation in mesenchymal tumors.

Labdane type diterpenes down-regulate the expression of c-Myc protein, but not of Bcl-2, in human leukemia T-cells undergoing apoptosis.

Sclareol (1) and ent-3beta-hydroxy-13-epi-manoyl oxide (2) belong to the labdane type diterpenes. They were isolated from the leaves and from the fruits of Cistus creticus subsp. creticus, and were found to be active against human leukemic cell lines. Compound 2 was converted to its thiomidazolide derivative (3). Compounds 1 and 3 were found to induce apoptotic cell death in human T-cell leukemia lines and to interfere with their cell cycle, arresting cells at G(0/1) phase. Apoptosis can involve the activation and/or suppression of critical genes such as c-myc whose reduction or its inappropriate expression can be associated with induction of cell death and bcl-2 whose activation prevents apoptosis in the latter case. In order to detect any concomitant effect (1 and 3) upon c-myc and bcl-2 oncogene expression, we performed Western blot analysis to determine the levels of expression of these two genes upon treatment with the above compounds. Western blot analysis showed that of c-myc proto-oncogene levels were markedly reduced before massive apoptosis ensued in H33AJ-JA1 and MOLT3 cells, while bcl-2 expression remained unaffected. Thus, induction of apoptosis due to compounds 1 and 3 in these T-cell leukemic cell lines is preceded by c-myc down regulation and furthermore sustained bcl-2 expression does not rescue cells from apoptosis under the conditions used.

The polyadenylation inhibitor cordycepin (3'dA) causes a decline in c-MYC mRNA levels without affecting c-MYC protein levels.

Study of the distribution of the poly(A) tail length of c-myc mRNA in several cell lines revealed a distinct, prevailing population with short poly(A) tails, derived through sequential deadenylation. To elucidate the possible in vivo function of this distinct short tailed c-myc mRNA population, the polyadenylation inhibitor cordycepin was used. This resulted in a decline in steady state c-myc mRNA levels with the remaining messenger mostly oligoadenylated. However, c-MYC proteins did not follow the reduction of the c-myc mRNA. On the other hand, in cells exposed to physiological agents known to downregulate c-myc expression, the reduction of mRNA steady state levels, was reflected upon c-MYC protein levels. The dissociation between c-myc mRNA and protein levels caused by cordycepin was not due to the stabilization of the c-MYC proteins and was not an indiscriminate effect since in the presence of cordycepin, c-fos mRNA and protein levels concomitantly declined. Our data indicate that under these conditions, a long poly(A) tail is not instrumental for c-myc mRNA translation and furthermore, the discrepancy in the steady state of c-myc mRNA level: c-MYC protein ratio between control cells and cells treated with cordycepin indicates that c-myc mRNA is subjected to translational control.