Electron microscopy reveals toroidal shape of master neuronal cell differentiator REST - RE1-silencing transcription factor.

The RE1-Silencing Transcription factor (REST) is essential for neuronal differentiation. Here, we report the first 18.5-angstrom electron microscopy structure of human REST. The refined electron map suggests that REST forms a torus that can accommodate DNA double-helix in the central hole. Additionally, we quantitatively described REST binding to the canonical DNA sequence of the neuron-restrictive silencer element. We developed protocols for the expression and purification of full-length REST and the shortened variant REST-N62 produced by alternative splicing. We tested the mutual interaction of full-length REST and the splicing variant REST-N62. Revealed structure-function relationships of master neuronal repressor REST will allow finding new biological ways of prevention and treatment of neurodegenerative disorders and diseases.

Inactivation of SARS-CoV-2 by charged particles for Future Vaccine Production Applications: A Monte Carlo study.

The world is still suffering from the SARS-CoV-2 pandemic, and the number of infected people is still growing in many countries in 2022. Although great strides have been made to produce effective vaccines, efforts in this field should be accelerated, particularly due to the emergence of new variants. Using inactivated viruses is a conventional method of vaccine production. High levels of ionizing radiation can effectively inactivate viruses. Recently, studies on SARS-CoV-2 irradiation using low-LET radiations (e.g., gamma rays) have been performed. However, there are insufficient studies on the impact of charged particles on the inactivation of this virus. In this study, a realistic structure of SARS-CoV-2 is simulated by using Geant4 Monte Carlo toolkit, and the effect of electrons, protons, alphas, C-12, and Fe-56 ions on the inactivation of SARS-CoV-2 is investigated. The simulation results indicated that densely ionizing (high-LET) particles have the advantage of minimum number of damaged spike proteins per single RNA break. The RNA breaks induced by hydroxyl radicals produced in the surrounding water medium were significant only for electron beam radiation. Hence, indirect RNA breaks induced by densely ionizing particles is negligible. From a simulation standpoint, alpha particles (with energies up to 30 MeV) as well as C-12 ions (with energies up to 80 MeV/n), and Fe-56 ions (with any energy) can be introduced as particles of choice for effective SARS-CoV-2 inactivation.

First high-quality genome assembly data of sago palm (Metroxylon sagu Rottboll).

The sago palm (Metroxylon sagu Rottboll) is a tropical halophytic starch-producing, economically important crop palm mainly located in Southeast Asian countries. Recently, a genome survey was conducted on this palm using the Illumina sequencing platform, with a very low (21.5%) BUSCO genome completeness score, and most of them (∼78%) are either fragmented or missing. Thus, in this study, the sago palm genome completeness was further improved with the utilization of the Nanopore sequencing platform that produced longer reads. A hybrid genome assembly was conducted, and the outcome was a much complete sago palm genome with BUSCO completeness achieved at as high as 97.9%, with only ∼2% of them either fragmented or missing. The estimated genome size of the sago palm is 509,812,790 bp in this study. A sum of 33,242 protein-coding genes was revealed from the sago palm genome and around 96.39% of them had been functionally annotated. An investigation on the carbohydrate metabolism KEGG pathways also unearthed that starch synthesis was one of the major sago palm activities. The genome data obtained from this work is indispensable for future molecular evolutionary and genome-wide association studies on the economically important sago palm.

Two SMARCAD1 Variants Causing Basan Syndrome in a Canadian and a Dutch Family.

Basan syndrome is an autosomal dominant genodermatosis characterized by congenital adermatoglyphia, transient congenital facial milia, neonatal acral bullae, and absent or reduced sweating. Basan syndrome is rare and has been reported in only 10 kindreds worldwide. It is caused by variants in the skin-specific isoform of SMARCAD1, which starts with an alternative exon 1. All reported variants, except for one large deletion, are point mutations within the donor splice site of the alternative exon 1. In this paper, we report two families with Basan syndrome and describe two SMARCAD1 variants. In one family, we have identified a complex structural variant (a deletion and a nontandem inverted duplication) using whole-genome optical mapping and whole-genome sequencing. Although this variant results in the removal of the first nine exons of SMARCAD1 and exon 1 of the skin-specific isoform, it manifested in the typical Basan phenotype. This suggests that unlike the skin-specific isoform, a single copy of full-length SMARCAD1 is sufficient for its respective function. In the second family, whole-exome sequencing revealed a deletion of 12 base pairs spanning the exon‒intron junction of the alternative exon 1 of the skin-specific SMARCAD1 isoform. In conclusion, we report two additional families with Basan syndrome and describe two SMARCAD1 pathogenic variants.

Genetic Risk Stratification: A Paradigm Shift in Prevention of Coronary Artery Disease.

Coronary artery disease (CAD) is a pandemic disease that is highly preventable as shown by secondary prevention. Primary prevention is preferred knowing that 50% of the population can expect a cardiac event in their lifetime. Risk stratification for primary prevention using the American Heart Association/American College of Cardiology predicted 10-year risk based on conventional risk factors for CAD is less than optimal. Conventional risk factors such as hypertension, cholesterol, and age are age-dependent and not present until the sixth or seventh decade of life. The genetic risk score (GRS), which is estimated from the recently discovered genetic variants predisposed to CAD, offers a potential solution to this dilemma. The GRS, which is derived from genotyping the population with a microarray containing these genetic risk variants, has indicated that genetic risk stratification based on the GRS is superior to that of conventional risk factors in detecting those at high risk and who would benefit most from statin therapy. Studies performed in >1 million individuals confirmed genetic risk stratification is superior and primarily independent of conventional risk factors. Prospective clinical trials based on risk stratification for CAD using the GRS have shown lifestyle changes, physical activity, and statin therapy are associated with 40% to 50% reduction in cardiac events in the high genetic risk group (20%). Genetic risk stratification has the advantage of being innate to an individual's DNA, and because DNA does not change in a lifetime, it is independent of age. Genetic risk stratification is inexpensive and can be performed worldwide, providing risk analysis at any age and thus has the potential to revolutionize primary prevention.

Hypertension and the roles of the 9p21.3 risk locus: Classic findings and new association data.

BACKGROUND: The band 9p21.3 contains an established genomic risk zone for cardiovascular disease (CVD). Since the initial 2007 Wellcome Trust Case Control Consortium study (WTCCC), the increased CVD risk associated with 9p21.3 has been confirmed by multiple studies in different continents. However, many years later there was still no confirmed report of a corresponding association of 9p21.3 with hypertension, a major CV risk factor, nor with blood pressure (BP). THEORY: In this contribution, we review the bipartite haplotype structure of the 9p21.3 risk locus: one block is devoid of protein-coding genes but contains the lead CVD risk SNPs, while the other block contains the first exon and regulatory DNA of the gene for the cell cycle inhibitor p15. We consider how findings from molecular biology offer possibilities of an involvement of p15 in hypertension etiology, with expression of the p15 gene modulated by genetic variation from within the 9p21.3 risk locus. RESULTS: We present original results from a Colombian study revealing moderate but persistent association signals for BP and hypertension within the classic 9p21.3 CVD risk locus. These SNPs are mostly confined to a 'hypertension island' that spans less than 60 kb and coincides with the p15 haplotype block. We find confirmation in data originating from much larger, recent European BP studies, albeit with opposite effect directions. CONCLUSION: Although more work will be needed to elucidate possible mechanisms, previous findings and new data prompt reconsidering the question of how variation in 9p21.3 might influence hypertension components of cardiovascular risk.

Good or not good: Role of miR-18a in cancer biology.

miR-18a is a member of primary transcript called miR-17-92a (C13orf25 or MIR17HG) which also contains five other miRNAs: miR-17, miR-19a, miR-20a, miR-19b and miR-92a. This cluster as a whole shows specific characteristics, where miR-18a seems to be unique. In contrast to the other members, the expression of miR-18a is additionally controlled and probably functions as its own internal controller of the cluster. miR-18a regulates many genes involved in proliferation, cell cycle, apoptosis, response to different kinds of stress, autophagy and differentiation. The disturbances of miR-18a expression are observed in cancer as well as in different diseases or pathological states. The miR-17-92a cluster is commonly described as oncogenic and it is known as 'oncomiR-1', but this statement is a simplification because miR-18a can act both as an oncogene and a suppressor. In this review we summarize the current knowledge about miR-18a focusing on its regulation, role in cancer biology and utility as a potential biomarker.

Advances in optical mapping for genomic research.

Recent advances in optical mapping have allowed the construction of improved genome assemblies with greater contiguity. Optical mapping also enables genome comparison and identification of large-scale structural variations. Association of these large-scale genomic features with biological functions is an important goal in plant and animal breeding and in medical research. Optical mapping has also been used in microbiology and still plays an important role in strain typing and epidemiological studies. Here, we review the development of optical mapping in recent decades to illustrate its importance in genomic research. We detail its applications and algorithms to show its specific advantages. Finally, we discuss the challenges required to facilitate the optimization of optical mapping and improve its future development and application.

Long walk to genomics: History and current approaches to genome sequencing and assembly.

Genomes represent the starting point of genetic studies. Since the discovery of DNA structure, scientists have devoted great efforts to determine their sequence in an exact way. In this review we provide a comprehensive historical background of the improvements in DNA sequencing technologies that have accompanied the major milestones in genome sequencing and assembly, ranging from early sequencing methods to Next-Generation Sequencing platforms. We then focus on the advantages and challenges of the current technologies and approaches, collectively known as Third Generation Sequencing. As these technical advancements have been accompanied by progress in analytical methods, we also review the bioinformatic tools currently employed in de novo genome assembly, as well as some applications of Third Generation Sequencing technologies and high-quality reference genomes.

Cardiac-Referenced Leukocyte Telomere Length and Outcomes After Cardiovascular Surgery.

Leukocyte telomere shortening reflects stress burdens and has been associated with cardiac events. However, the patient-specific clinical value of telomere assessment remains unknown. Moreover, telomere shortening cannot be inferred from a single telomere length assessment. The authors investigated and developed a novel strategy for gauging leukocyte telomere shortening using autologous cardiac atrial referencing. Using multitissue assessments from 163 patients who underwent cardiovascular surgery, we determined that the cardiac atrium-leukocyte telomere length difference predicted post-operative complexity. This constituted the first evidence that a single-time assessment of telomere dynamics might be salient to acute cardiac care.

Identification of Positively and Negatively Selected Driver Gene Mutations Associated With Colorectal Cancer With Microsatellite Instability.

Background & Aims: Recent studies have shown that cancers arise as a result of the positive selection of driver somatic events in tumor DNA, with negative selection playing only a minor role, if any. However, these investigations were concerned with alterations at nonrepetitive sequences and did not take into account mutations in repetitive sequences that have very high pathophysiological relevance in the tumors showing microsatellite instability (MSI) resulting from mismatch repair deficiency investigated in the present study. Methods: We performed whole-exome sequencing of 47 MSI colorectal cancers (CRCs) and confirmed results in an independent cohort of 53 MSI CRCs. We used a probabilistic model of mutational events within microsatellites, while adapting pre-existing models to analyze nonrepetitive DNA sequences. Negatively selected coding alterations in MSI CRCs were investigated for their functional and clinical impact in CRC cell lines and in a third cohort of 164 MSI CRC patients. Results: Both positive and negative selection of somatic mutations in DNA repeats was observed, leading us to identify the expected true driver genes associated with the MSI-driven tumorigenic process. Several coding negatively selected MSI-related mutational events (n = 5) were shown to have deleterious effects on tumor cells. In the tumors in which deleterious MSI mutations were observed despite the negative selection, they were associated with worse survival in MSI CRC patients (hazard ratio, 3; 95% CI, 1.1-7.9; P = .03), suggesting their anticancer impact should be offset by other as yet unknown oncogenic processes that contribute to a poor prognosis. Conclusions: The present results identify the positive and negative driver somatic mutations acting in MSI-driven tumorigenesis, suggesting that genomic instability in MSI CRC plays a dual role in achieving tumor cell transformation. Exome sequencing data have been deposited in the European genome-phenome archive (accession: EGAS00001002477).

Functional redundancy in Echinocandin B in-cluster transcription factor ecdB of Emericella rugulosa NRRL 11440.

Echinocandin B is a potent antifungal against the majority of fungal pathogens and its biosynthesis occurred by ecd and hty gene clusters in Emericella rugulosa NRRL 11440. We elucidated the functional necessity of in-clustered transcription factor; ecdB in the production of echinocandin B. We deleted the ecdB gene and found that ΔecdB mutant has no significant effect on echinocandin B production. The expression level of most of the ecd and hty cluster genes was not significantly altered except few of them up-regulated in knockout strain. The complete abrogation in ecdB gene expression was observed in ΔecdB strain. However, the interactions of purified EcdB protein with DNA sequence of ecdA, ecdH, ecdK and ecdI promoter was confirmed in-vitro. Our results conclude that EcdB protein in-vitro binds to the ecdA, ecdH, ecdK and ecdI promoter but in-vivo, it could not significantly affect the gene expression and echinocandin B production in Emericella rugulosa.

Ferroportin Expression in Adipocytes Does Not Contribute to Iron Homeostasis or Metabolic Responses to a High Calorie Diet.

BACKGROUND & AIMS: Iron has an increasingly recognized role in the regulation of adipose tissue function, including the expression of adipokines involved in the pathogenesis of nonalcoholic fatty liver disease. The cellular iron exporter, ferroportin, has been proposed as being a key determinant of adipocyte iron homeostasis. METHODS: We studied an adipocyte-specific ferroportin (Fpn1) knockout mouse model, using an Adipoq-Cre recombinase driven Fpn1 deletion and fed mice according to the fast food diet model of nonalcoholic steatohepatitis. RESULTS: We showed successful selective deletion of Fpn1 in adipocytes, but found that this did not lead to increased adipocyte iron stores as measured by atomic absorption spectroscopy or histologically quantified iron granules after staining with 3,3'-diaminobenzidine-enhanced Perls' stain. Mice with adipocyte-specific Fpn1 deletion did not show dysregulation of adiponectin, leptin, resistin, or retinol-binding protein-4 expression. Similarly, adipocyte-specific Fpn1 deletion did not affect insulin sensitivity during hyperinsulinemic-euglycemic clamp studies or lead to histologic evidence of increased liver injury. We have shown, however, that the fast food diet model of nonalcoholic steatohepatitis generates an increase in adipose tissue macrophage infiltration with crown-like structures, as seen in human beings, further validating the utility of this model. CONCLUSIONS: Ferroportin may not be a key determinant of adipocyte iron homeostasis in this knockout model. Further studies are needed to determine the mechanisms of iron metabolism in adipocytes and adipose tissue.

High level expression and purification of recombinant flounder growth hormone in E. coli.

Recombinant flounder growth hormone was overproduced in E. coli by using codon optimized synthetic gene and optimized expression conditions for high level production. The gene was cloned into PET-28a expression vector and transformed into E. coli BL21 (DE3). Induction at lower temperature, lower IPTG concentrations and richer growth media during expression resulted in increased expression level. The protein expression profile was analyzed by SDS-PAGE, the authenticity was confirmed by western blotting and the concentration was determined by Bradford assay. In addition, several attempts were made to produce soluble product and all resulted in insoluble product. The overexpressed protein was efficiently purified from inclusion bodies by moderate speed centrifugation after cell lysis. Among the solubilization buffers examined, buffer with 1% N-lauroylsarcosine in the presence of reducing agent DTT at alkaline pH resulted in efficient solubilization and recovery. The denaturant was removed by filtration and dialysis. The amount of the growth hormone recovered was significantly higher than previous reports that expressed native growth hormone genes in E. coli. The methodology adapted in this study, can be used to produce flounder growth hormone at large scale level so that it can be used in aquaculture. This approach may also apply to other proteins if high level expression and efficient purification is sought in E. coli.

GATA4 Is Sufficient to Establish Jejunal Versus Ileal Identity in the Small Intestine.

BACKGROUND & AIMS: Patterning of the small intestinal epithelium along its cephalocaudal axis establishes three functionally distinct regions: duodenum, jejunum, and ileum. Efficient nutrient assimilation and growth depend on the proper spatial patterning of specialized digestive and absorptive functions performed by duodenal, jejunal, and ileal enterocytes. When enterocyte function is disrupted by disease or injury, intestinal failure can occur. One approach to alleviate intestinal failure would be to restore lost enterocyte functions. The molecular mechanisms determining regionally defined enterocyte functions, however, are poorly delineated. We previously showed that GATA binding protein 4 (GATA4) is essential to define jejunal enterocytes. The goal of this study was to test the hypothesis that GATA4 is sufficient to confer jejunal identity within the intestinal epithelium. METHODS: To test this hypothesis, we generated a novel Gata4 conditional knock-in mouse line and expressed GATA4 in the ileum, where it is absent. RESULTS: We found that GATA4-expressing ileum lost ileal identity. The global gene expression profile of GATA4-expressing ileal epithelium aligned more closely with jejunum and duodenum rather than ileum. Focusing on jejunal vs ileal identity, we defined sets of jejunal and ileal genes likely to be regulated directly by GATA4 to suppress ileal identity and promote jejunal identity. Furthermore, our study implicates GATA4 as a transcriptional repressor of fibroblast growth factor 15 (Fgf15), which encodes an enterokine that has been implicated in an increasing number of human diseases. CONCLUSIONS: Overall, this study refines our understanding of an important GATA4-dependent molecular mechanism to pattern the intestinal epithelium along its cephalocaudal axis by elaborating on GATA4's function as a crucial dominant molecular determinant of jejunal enterocyte identity. Microarray data from this study have been deposited into NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) and are accessible through GEO series accession number GSE75870.

Interdependency of EGF and GLP-2 Signaling in Attenuating Mucosal Atrophy in a Mouse Model of Parenteral Nutrition.

BACKGROUND & AIMS: Total parenteral nutrition (TPN), a crucial treatment for patients who cannot receive enteral nutrition, is associated with mucosal atrophy, barrier dysfunction, and infectious complications. Glucagon-like peptide-2 (GLP-2) and epidermal growth factor (EGF) improve intestinal epithelial cell (IEC) responses and attenuate mucosal atrophy in several TPN models. However, it remains unclear whether these 2 factors use distinct or overlapping signaling pathways to improve IEC responses. We investigated the interaction of GLP-2 and EGF signaling in a mouse TPN model and in patients deprived of enteral nutrition. METHODS: Adult C57BL/6J, IEC-Egfrknock out (KO) and IEC-pik3r1KO mice receiving TPN or enteral nutrition were treated with EGF or GLP-2 alone or in combination with reciprocal receptor inhibitors, GLP-2(3-33) or gefitinib. Jejunum was collected and mucosal atrophy and IEC responses were assessed by histologic, gene, and protein expression analyses. In patients undergoing planned looped ileostomies, fed and unfed ileum was analyzed. RESULTS: Enteral nutrient deprivation reduced endogenous EGF and GLP-2 signaling in mice and human beings. In the mouse TPN model, exogenous EGF or GLP-2 attenuated mucosal atrophy and restored IEC proliferation. The beneficial effects of EGF and GLP-2 were decreased upon Gefitinib treatment and in TPN-treated IEC-EgfrKO mice, showing epidermal growth factor-receptor dependency for these IEC responses. By contrast, in TPN-treated IEC-pi3kr1KO mice, the beneficial actions of EGF were lost, although GLP-2 still attenuated mucosal atrophy. CONCLUSIONS: Upon enteral nutrient deprivation, exogenous GLP-2 and EGF show strong interdependency for improving IEC responses. Understanding the differential requirements for phosphatidylinositol 3-kinase/phosphoAKT (Ser473) signaling may help improve future therapies to prevent mucosal atrophy.

Biochemical and molecular modulation of CCl4-induced peripheral and central damage by Tilia americana var. mexicanaextracts.

Around the world, species from the genus Tilia are commonly used because of their peripheral and central medicinal effects; they are prepared as teas and used as tranquilizing, anticonvulsant, and analgesic agents. In this study, we provide evidence of the protective effects of organic and aqueous extracts (100 mg/kg, i.p.) obtained from the leaves of Tilia americana var. mexicana on CCl4-induced liver and brain damage in the rat. Protection was observed in the liver and brain (cerebellum, cortex and cerebral hemispheres) by measuring the activity of antioxidant enzymes and levels of malondialdehyde (MDA) using spectrophotometric methods. Biochemical parameters were also assessed in serum samples from the CCl4-treated rats. The T. americana var. mexicana leaf extracts provided significant protection against CCl4-induced peripheral and central damage by increasing the activity of antioxidant enzymes, diminishing lipid peroxidation, and preventing alterations in biochemical serum parameters, such as the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ-globulin (γ-GLOB), serum albumin (ALB), total bilirubin (BB), creatinine (CREA) and creatine kinase (CK), relative to the control group. Additionally, we correlated gene expression with antioxidant activity in the experimental groups treated with the organic and aqueous Tilia extracts and observed a non-statistically significant positive correlation. Our results provide evidence of the underlying biomedical properties of T. americana var. mexicana that confer its neuro- and hepatoprotective effects.

Molecular techniques for the personalised management of patients with chronic myeloid leukaemia.

Chronic myeloid leukemia (CML) is the paradigm for targeted cancer therapy. RT-qPCR is the gold standard for monitoring response to tyrosine kinase-inhibitor (TKI) therapy based on the reduction of blood or bone marrow BCR-ABL1. Some patients with CML and very low or undetectable levels of BCR-ABL1 transcripts can stop TKI-therapy without CML recurrence. However, about 60 percent of patients discontinuing TKI-therapy have rapid leukaemia recurrence. This has increased the need for more sensitive and specific techniques to measure residual CML cells. The clinical challenge is to determine when it is safe to stop TKI-therapy. In this review we describe and critically evaluate the current state of CML clinical management, different technologies used to monitor measurable residual disease (MRD) focus on comparingRT-qPCR and new methods entering clinical practice. We discuss advantages and disadvantages of new methods.

Genetic variation in wild and hatchery populations of giant freshwater prawn (Macrobrachium rosenbergii) revealed by randomly amplified polymorphic DNA markers.

Sustainable improvement and conservation of any genetic resource depend on the assessment of its intra- and inter-population genetic variation. In order to estimate genetic variation in both wild and hatchery populations of Macrobrachium rosenbergii, randomly amplified polymorphic DNA (RAPD) analysis was performed. Analyses of 51 polymorphic loci amplified from genomic DNA by three decamer random primers revealed different degrees of genetic variation in two wild (Bhairab and Rupsha rivers) and hatchery-derived gher (Gher-1 and Gher-2) populations. The proportion of polymorphic loci was found to be higher in wild populations (0.90 and 0.65 for the Bhairab and Rupsha populations, respectively) than the hatchery-derived gher populations (0.29 and 0.16 for Gher-1 and Gher-2, respectively). Likewise, the river populations contained higher levels of gene diversity (0.221 and 0.179 for Bhairab and Rupsha populations, respectively) than the gher populations (0.114 and 0.045 for Gher-1 and Gher-2, respectively). These results suggest reduction of genetic variation and heterozygosity in the hatchery-derived gher populations. Inter-population similarity indices and pairwise genetic distance values showed that variation between the wild or between the gher populations were lower than those between the wild and hatchery populations. On average, 14 loci exhibited significant deviation from homogeneity in wild vs hatchery population pairs, whereas 2 and 3 loci showed heterogeneity in Gher-1 vs Gher-2 and Bhairab vs Rupsha population pairs, respectively. A genetic distance-based UPGMA dendrogram segregated river populations from the gher populations. Our study, therefore, revealed substantial levels of genetic variation between wild and hatchery populations of M. rosenbergii.

Detection, molecular typing and phylogenetic analysis of Leishmania isolated from cases of leishmaniasis among Syrian refugees in Lebanon.

Leishmania is a parasitic protozoan with more than two-dozen species causing the disease leishmaniasis. It is transmitted to humans through the bite of an infected female phlebotomine sand-fly vector. In the past two years the incidence of leishmaniasis has been drastically increasing in Lebanon. This was in parallel with the deterioration of the security in Syria forcing thousands to flee and seek shelter in poorly maintained refugee camps and collective shelters. Cutaneous leishmaniasis (CL) is now considered a public health problem, but its epidemiology has not been fully elucidated. To our knowledge, this is the first study comparing two different molecular methods for the detection and identification of Leishmania tropica in Lebanon. Two molecular typing methods of 39 FFPE Leishmania isolates were used: the ITS1-PCR RFLP and the nested ITS1-5.8S rDNA gene amplification followed by sequencing and phylogenetic analysis. The efficiency of these two techniques in Leishmania identification was compared and the phylogenetic relationships among these isolates were illustrated based on the neighbor-joining (NJ) method. The results were statistically correlated with the parasitic index (PI). The DNA storage in formalin-fixed paraffin embedded (FFPE) tissues was assessed as well. The parasites identified were all L. tropica as determined by both techniques. ITS1-5.8S rDNA gene based typing proved to be more sensitive in the detection of parasites (positive in 69.2% of the isolates) as opposed to the ITS1-PCR RFLP method that was successful in identifying L. tropica in only 43.6% of the isolates. Sequencing and phylogenetic analysis revealed high levels of heterogeneity. A statistically significant correlation was observed between PI and the results of the nested ITS1-5.8S rDNA gene PCR. Genotyping at the species level is essential for monitoring the relative frequency of CL in the Mediterranean area that is correlated to three different Leishmania species (Leishmania infantum, Leishmania major and L. tropica), each characterized by distinct epidemiological features. The obtained results highlight the need to find a universally accepted diagnostic tool for Leishmania typing.