Valentina Nikolic

mTOR-independent autophagy counteracts apoptosis in herpes simplex virus type 1-infected U251 glioma cells.

We investigated the role of autophagy, a stress-inducible lysosomal self-digestion of cellular components, in modulation of herpes simplex virus type 1 (HSV-1)-triggered death of U251 human glioma cells. HSV-1 caused apoptotic death in U251 cells, characterized by phosphatidylserine externalization, caspase activation and DNA fragmentation. HSV-1-induced apoptosis was associated with the induction of autophagic response, as confirmed by the conversion of cytosolic LC3-I to autophagosome-associated LC3-II, increase in intracellular acidification, presence of autophagic vesicles, and increase in proteolysis of the selective autophagic target p62. HSV-1-triggered autophagy was not associated with the significant increase in the expression of proautophagic protein beclin-1 or downregulation of the major autophagy suppressor mammalian target of rapamycin (mTOR). Moreover, the phosphorylation of mTOR and its direct substrate p70 S6 kinase was augmented by HSV-1 infection, while the mTOR stimulator Akt and inhibitor AMPK-activated protein kinase (AMPK) were accordingly activated and suppressed, respectively. An shRNA-mediated knockdown of the autophagy-essential LC3β, as well as pharmacological inhibition of autophagy with bafilomycin A1 or 3-methyladenine, markedly accelerated apoptotic changes and ensuing cell death in HSV-1-infected glioma cells. These data indicate that AMPK/Akt/mTOR-independent autophagy could prolong survival of HSV-1-infected U251 glioma cells by counteracting the coinciding apoptotic response.

Evidence of recombination in Tula virus strains from Serbia.

Tula hantavirus (TULV) belongs to Bunyaviridae family, with negative sense RNA genome. Segmented nature of hantaviral genome allows for genetic reassortment, but the evidence of homologous recombination also exists. In this study we analyzed TULV sequences isolated in Serbia on different occasions and from different rodent hosts: 1987 strain from Microtus subterraneus and 2007 strain from Microtus arvalis. Phylogenetic analysis of both L and S segment sequences is suggestive of geographically related clustering, as previously shown for majority of hantaviruses. Reconstruction of phylogenetic tree for TULV S segment showed that both sequences from Serbia clustered together with sequences from East Slovakia, which had previously been shown to be recombinants (Kosice strain). Exploratory recombination analysis, supported by phylogenetic and amino acid pattern analysis, revealed the presence of recombination in the S segment sequences from Serbia, resulting in mosaic-like structure of TULV S segment similar to the one of Kosice strain. Although recombination is considered a rare event in molecular evolution of negative strand RNA viruses, obtained molecular data in this study support evidence of recombination in TULV, in geographically distant regions of Europe.

Molecular typing of the local HIV-1 epidemic in Serbia.

Worldwide HIV-1 pandemic is becoming increasingly complex, with growing heterogeneity of subtypes and recombinant viruses. Previous studies have documented HIV-1 subtype B as the predominant one in Serbia, with limited presence and genetic diversity of non B subtypes. In recent years, MSM transmission has become the most frequently reported risk for HIV infection among newly diagnosed patients in Serbia, but very little is known of the network structure and dynamics of viral transmission in this and other risk groups. To gain insight about the HIV-1 subtypes distribution pattern as well as characteristics of HIV-1 transmission clusters in Serbia, we analyzed the genetic diversity of the pol gene segment in 221 HIV-1-infected patients sampled during 2002-2011. Subtype B was found to still be the most prevalent one in Serbia, accounting for over 90% of samples, while greater diversity of other subtypes was found than previously reported, including subtypes G, C, A, F, CRF01 and CRF02. In total, 41.3% of analyzed subtype B sequences were found associated in transmission clusters/network, that are highly related with MSM transmission route.

The Third Branch of the Main Trunk of the Left Coronary Artery in Cercopithecus aethiops sabaeus. Is the Nonhuman Primate Model Appropriate

The aim of this study was to investigate the morphometry of branching patterns of the main trunk of the left coronary artery (MT of LCA) in nonhuman primates, and comment on the current nomenclature. The biometric study was performed using stereomicroscopic dissection of hearts of healthy and fertile nonhuman primates (Cercopithecus aethiops sabaeus) of both sexes. Our results reveal that the MT of LCA terminates in a bifurcation into the anterior interventricular branch (AIB) and the circumflex branch (CB) (74.6%), trifurcation into the AIB, CB, and diagonal branch (DB) (23.6%), or occasionally quadrifurcation into the AIB, CB, and two DBs (1.8%). This is similar to the case in humans. Furthermore, two morphological aspects of the DB spatial distribution, in addition to its branching pattern, resemble the DB in humans. Myocardial bridges observed over the DB in the Cercopithecus aethiops heart further contribute to the similarity with humans. The resemblance of the DB and its branches to their human counterparts make them a suitable model for experimental study on coronary circulation. Anat Rec, 2011.

Hamstring Regeneration Following Harvest for Anterior Cruciate Ligament Reconstruction

The semitendinosus (ST) and gracillis (Gr) tendons are commonly used as a replacement graft during the anterior cruciate ligament (ACL) reconstruction. There are many advantages of using the hamstrings tendons, including the ease of harvesting, suitable morphology for use as a graft, lower donor site morbidity, early rehabilitation, and patient satisfaction.

Decapitation in reality and fine art: A review

The aim of our study was to examine all types of decapitation from forensic literature, including our own case, and to analyze the presentation of beheading in fine art, popular literature, and music. To do this, over 200 scientific articles in regard to decapitation were analyzed, as well as more than 10,000 artworks, and several hundreds of literary works and music pieces. In addition, a macroscopic examination of a decapitated victim was performed. Finally, a multislice computerized tomography (MSCT) examination of the cervical spine in two live volunteers was undertaken to present the osteological relationships. The forensic and criminal investigation revealed that a female victim was murdered by her jealous husband by applying several strikes with an axe, which resulted in an incomplete decapitation. All the main neck structures were transected, including the cervical spine, except a smaller part of the skin and soft tissue in the nuchal region. The mentioned MSCT examination in both the neutral position and flexion showed that the mandible can also be injured in a higher cervical location of the severance line. Various types of beheading were mentioned, including a homicidal, suicidal, accidental, judicial, internal, pathophysiological, and foetal ones. The status of consciousness and emotions in individuals just before and after decapitation was discussed. Finally, it was found that decapitation was the subject of many artists, and some writers and musicians. In conclusion, we presented a rare case of a homicide beheading performed with an axe. In addition, forensic importance of decapitation was discussed, as well as its great medical, social, anthropological, and artistic significance.

Locally advanced rectal cancers with simultaneous occurrence of KRAS mutation and high VEGF expression show invasive characteristics

In this study, we investigated the mutation status of KRAS gene in pretherapeutic and preoperative biopsies in 63 specimens of locally advanced rectal cancers in order to evaluate its potential predictive and/or prognostic role. Regions of interest of KRAS exon 2 were amplified and visualized on 2% agarose gel. Obtained PCR products were subjected to direct sequencing. KRAS mutations were detected in 35% of patients, 91% of which were located in codon 12 and 9% in codon 13. In general, KRAS mutation status did not affect the response to neoadjuvant chemoradiotherapy (CRT). However, patients harboring mutated KRAS gene, simultaneously with high vascular endothelial growth factor (VEGF) expression, exhibited a worse response to CRT (p=0.030), a more frequent appearance of local recurrences and distant metastasis (p=0.003), and shorter overall survival (p=0.001) compared to all others. On the contrary, patients with GGT>GCT KRAS mutation exhibited a significantly better response to CRT than those with any other type of KRAS mutation (p=0.017). Moreover, the presence of GGT>GCT mutation was associated with low VEGF and Ki67 expression (p=0.012 in both cases), parameters related to less aggressiveness of the disease. Our results suggest that KRAS mutation status could have some predictive and prognostic importance in rectal cancer when analyzed together with other parameters, such as VEGF and Ki67 expression. In addition, it seems that not only the presence but the type of KRAS mutation is important for examining its impact on CRT response.

An Extremely Rare Coronary Variation: Direct Communication between the Circumflex and Right Coronary Arteries.

Coronary collateral circulation frequently develops in the presence of obstructive coronary lesions as an alternative source of blood supply. We present a case of rare direct communication between the circumflex and right coronary arteries in a 30-year-old male with continuity between the distal segments of the circumflex and right coronary artery and no coronary artery stenoses. Direct intercoronary connections may be seen in the absence of an occlusive coronary lesion, as a remnant of the fetal circulation in adult life. We present this case to increase awareness of this rare coronary variation and its clinical implications.

Forensic application of phylogenetic analysis – exploration of suspected epidemiological linkage

Phylogenetic analysis may serve as a valuable tool in assessing the epidemiological relation between viral DNA sequences. In order to increase the likelihood of observing phylogenetic separation of sequences as well as to give strong forensic evidence regarding transmission, phylogenetic analysis needs to be performed on appropriate local control sequences and by sequencing of at least two genetic regions of reasonable length, depending on the gene under investigation. The aim of this study was to explore the suspected epidemiological linkage between DNA sequences isolated from three HIV-1 infected patients by means of phylogenetic analyses. Phylogenetic analysis was performed on investigated sequences together with a number of local controls, that are viral sequences from infected individuals in the same location and diagnosed in a similar time period. Two genetic regions, of 1,6 kb for pol and 800 bp for env, were amplified and sequenced from viral RNA extracted from plasma. Transmission clusters were assigned as those phylogenetic clades consisting of three or more sequences, fulfilling the conditions of genetic distance of 1.5% or less with posterior probability higher than 0.9 in the Bayesian analyses, for both genetic region. Maximum likelihood (ML) phylogenetic analysis was performed as implemented in Phylogenetic Analysis Using Parsimony (PAUP), using the evolutionary model selected by jModeltest software. Phylogenetic analysis revealed the presence of a single transmission cluster that accomplished all predefined sets of criteria. This cluster was composed of three viral sequences isolated from patients whose epidemiological linkage was under investigation. The mean pairwise nucleotide divergence among all observed pol sequences was 7.1% (range 0.2-12.8%) while among sequences under investigation it was 0.8% (0.3-1.1%). Regarding env sequences, nucleotide divergence among all was 16.1% (range 0.3-26.3%), while among sequences under investigation it was 1.3% (1.1-1.5%). Our results strongly support the epidemiological linkage between sequences under investigation. Viral phylogenies can help to evaluate proposed epidemiological linkage and to infer the ancestral relationships of infections. However phylogenetic analysis cannot unambiguously prove that HIV-1 transmission occurred directly between two individuals, since any transmission chain may contain additional sequences not included in the analysis.

The prevalence of RT 245 codon polymorphisms and its association with duration of infection among HIV-1 patients in Serbia

Amino acid (aa) substitutions at position 245 of HIV-1 reverse transcriptase (RT) have been described to be associated to the presence of human leukocyte antigen (HLA)–B*5701 allele in the host, in particular in subtype B infection. Preliminary data show HLA–B*5701 prevalence of around 6.5% in Serbian population. In this study, we investigated the prevalence of RT codon 245 substitutions among HIV infected patients in Serbia. Furthermore, we analyzed the association between RT 245 aa substitutions with duration of infection, estimated by proportion of ambiguous basecalls per sequence. The study included 184 consenting, subtype B HIV infected patients aged 18 or more. The majority of patients were newly diagnosed, 150/184, while 34/184 patients were on treatment. Pol region sequences, covering protease and minimally 250 RT codons, were obtained within routine drug resistance testing. The fraction of ambiguous nucleotides in each sequence was calculated for samples drawn from naive patients. We used ambiguity percentage of 0.47% as a cut-off value delimiting recent (less than 1 year) vs. chronic infection (longer than 1 year). In total, predominant aa at RT codon 245 was the wild type valin (V) found in 118/184 (64.1%), hence 35.9% (66/184) contained mutation at this position (among naive patients this percentage was 38% (57/150). The most common substitution at RT codon 245 was methionine (M) 29/184 (15.7%), followed by glutamic acid (E) 20/184 (10.9%) and others. Based on the percentage of ambiguous basecalls, a total of 55.3% of naive samples (83/150) were classified as recent infection, while among these, 57.8% (48/83) had V at position 245. A total of 45.3% (68/150) were classified as chronic infection, with the presence of V at RT codon 245 found in 69.1% (47/68). We did not find statistically significant association between polymorphisms at codon 245 and duration of infection (p=0.2080). The frequency of RT 245 substitution found in our study exceeds the estimated prevalence HLA–B*5701 in Serbian population. This may be related to the presence of other, similar HLA alleles, limiting specificity of the correlation between HLA-B*5701 and RT codon 245 variation. Furthermore, no statistically significant difference was found in the prevalence of RT 245 substitutions between recent and chronic infection. This may suggest early fixation of an HLA induced selective imprint, during viral evolution in an infected patient.