2021 European guideline on HIV testing in genito-urinary medicine settings.

Testing for HIV is critical for early diagnosis of HIV infection, providing long-term good health for the individual and prevention of onward transmission if antiretroviral treatment is initiated early. The main purpose of the 2021 European Guideline on HIV Testing in Genito-Urinary Settings is to provide advice on testing for HIV infection in individuals aged 16 years and older who present to sexually transmitted infection, genito-urinary or dermato-venereology clinics across Europe. The guideline presents the details of best practice and offers practical guidance to clinicians and laboratories to identify and offer HIV testing to appropriate patient groups.

Histone Modifications on the Promoters of Human OCT4 and NANOG Genes at the Onset of Neural Differentiation of NT2/D1 Cells.

Transcription factors OCT4 and NANOG are main constituents of a functional network that controls proliferation and pluripotency maintenance of stem cells as well as early lineage decisions. We investigated expression profiles of OCT4 and NANOG during the early phases of neural differentiation using NT2/D1 cells induced by retinoic acid as an in vitro model system of human neurogenesis. We demonstrated decrease in OCT4 and NANOG mRNA and protein levels following exposure to retinoic acid. Next, by employing chromatin immunoprecipitation, we investigated profiles of selected H3 and H2B histone marks deposited on the promoters of the OCT4 and NANOG genes. We found decline in H3K4me3, H2BK5ac, and H2BK120ac on both promoters, which paralleled the decrease in OCT4 and NANOG expression. Moreover, we found that the H2BK16ac mark is differentially enriched on these two promoters, pointing to differences in epigenetic regulation of OCT4 and NANOG gene expression. Finally, based on our data, we suggest that the early response of pluripotency genes OCT4 and NANOG to the differentiation-inducing stimuli is mediated by dynamic changes in chromatin marks, while DNA methylation is acquired in the later stages of neurogenesis.

Transcription factor NF-Y inhibits cell growth and decreases SOX2 expression in human embryonal carcinoma cell line NT2/D1.

Transcription factor NF-Y belongs to the embryonic stem cell transcription factor circuitry due to its role in the regulation of cell proliferation. We investigated the role of NF-Y in pluripotency maintenance using NT2/D1 cells as one of the best-characterized human embryonal carcinoma cell line. We investigated the efficiency of protein transduction and analyzed the effects of forced expression of short isoform of NF-Y A-subunit (NF-YAs) on NT2/D1 cell growth and expression of SOX2. We found that protein transduction is an efficient method for NF-Y overexpression in NT2/D1 cells. Next, we analyzed the effect of NF-YAs overexpression on NT2/D1 cell viability and detected significant reduction in cell growth. The negative effect of NF-YAs overexpression on NT2/D1 cell pluripotency maintenance was confirmed by the decrease in the level of the pluripotency marker SOX2. Finally, we checked the p53 status and determined that the NF-Y-induced inhibition of NT2/D1 cell growth is p53-independent.

EFFICACY OF METAMITRON IN APPLE THINNING IN SERBIA.

The thinning of fruits is a required pomotechnical measure in intensive fruit production which ensures the production of good quality fruits and high yields. Metamitron, known as inhibitor of photosynthesis, has been successfully used in the thinning of apple fruits. This study had the aim to determine the efficacy of metamitron on the thinning of apple fruits in the agroecological conditions of Serbia and to evaluate the possibility of its practical application. Two varieties of apples that are widely grown in Serbia, dared and Golden Delicious, have been chosen for this research. The experiments were carried out during 2011 and 2012 according to the EPPO PP 1/158 (3) method. Metamitron has shown a good efficacy in the thinning of apple fruits. The effect of metamitron on the thinning of apple fruits depends on multiple factors, pri- marily the application dose, time of application, apple variety, but also on the number of fruits developed. The best efficacy on the Idared variety was in plots where metamitron was applied at a dose of 1.1 kg ha⁻¹, once (in the growth stage when the fruits were 8 mm in diameter) or twice (in the growth stages when the fruits were 8 mm and 12 mm in diameter), when the number of developed fruits per tree is smaller, or 1.65 kg ha⁻¹ applied once when the fruits are 12 mm in size when a larger number of fruits per tree is developed. On the Golden Delicious variety, the best efficacy was in treatments when metamitron was applied twice (in the growth stages when the fruits were 8 mm and 12 mm in diameter) in quantities of 1.1 kg ha⁻¹, when less fruits per tree were formed or 1.65 kg ha⁻¹, applied once or twice when a larger number of fruits per tree were formed.

SOX2 overexpression affects neural differentiation of human pluripotent NT2/D1 cells.

SOX2 is one of the key transcription factors involved in maintenance of neural progenitor identity. However, its function during the process of neural differentiation, including phases of lineage-specification and terminal differentiation, is still poorly understood. Considering growing evidence indicating that SOX2 expression level must be tightly controlled for proper neural development, the aim of this research was to analyze the effects of constitutive SOX2 overexpression on outcome of retinoic acid-induced neural differentiation of pluripotent NT2/D1 cells. We demonstrated that in spite of constitutive SOX2 overexpression, NT2/D1 cells were able to reach final phases of neural differentiation yielding both neuronal and glial cells. However, SOX2 overexpression reduced the number of mature MAP2-positive neurons while no difference in the number of GFAP-positive astrocytes was detected. In-depth analysis at single-cell level showed that SOX2 downregulation was in correlation with both neuronal and glial phenotype acquisitions. Interestingly, while in mature neurons SOX2 was completely downregulated, astrocytes with low level of SOX2 expression were detected. Nevertheless, cells with high level of SOX2 expression were incapable of entering in either of two differentiation pathways, neurogenesis or gliogenesis. Accordingly, our results indicate that fine balance between undifferentiated state and neural differentiation depends on SOX2 expression level. Unlike neurons, astrocytes could maintain low level of SOX2 expression after they acquired glial fate. Further studies are needed to determine whether differences in the level of SOX2 expression in GFAP-positive astrocytes are in correlation with their self-renewal capacity, differentiation status, and/or their phenotypic characteristics.

Construction and functional analysis of novel dominant-negative mutant of human SOX18 protein.

SOX18 transcription factor plays important roles in a range of biological processes such as vasculogenesis, hair follicle development, lymphangiogenesis, atherosclerosis, and angiogenesis. In this paper we present the generation of a novel SOX18 dominant-negative mutant (SOX18DN) encoding truncated SOX18 protein that lacks a trans-activation domain. We show that both wild-type SOX18 (SOX18wt) and truncated human SOX18 proteins are able to bind to their consensus sequence in vitro. Functional analysis confirmed that SOX18wt has potent trans-activation properties, while SOX18DN displays dominant-negative effect. We believe that these SOX18wt and SOX18DN expression constructs could be successfully used for further characterization of the function of this protein.

Telomere-associated chromosome fragmentation: applications in genome manipulation and analysis.

Telomere-associated chromosome fragmentation (TACF) is a new approach for chromosome mapping based on the non-targeted introduction of cloned telomeres into mammalian cells. TACF has been used to generate a panel of somatic cell hybrids with nested terminal deletions of the long arm of the human X chromosome, extending from Xq26 to the centromere. This panel has been characterized using a series of X chromosome loci. Recovery of the end clones by plasmid rescue produces a telomeric marker for each cell line and partial sequencing will allow the generation of sequence tagged sites (STSs). TACF provides a powerful and widely applicable method for genome analysis, a general way of manipulating mammalian chromosomes and a first step towards constructing artificial mammalian chromosomes.

Establishment and initial characterization of SOX2-overexpressing NT2/D1 cell clones.

SOX2, a universal marker of pluripotent stem cells, is a transcription factor that helps control embryonic development in vertebrates; its expression persists in neural stem/progenitor cells into adulthood. Considering the critical role of the SOX2 transcription factor in the regulation of genes required for self-renewal and pluripotency of stem cells, we developed and characterized SOX2-overexpressing NT2/D1 cell clones. Using Southern blot and semi-quantitative RT-PCR, we confirmed integration and expression of exogenous SOX2 in three NT2/D1 cell clones. Overexpression of the SOX2 gene was detected in two of these clones. SOX2 overexpression in NT2/D1 cell clones resulted in altered expression of key pluripotency genes OCT4 and NANOG. Furthermore, SOX2-overexpressing NT2/D1 cell clones entered into retinoic acid-dependent neural differentiation, even when there was elevated SOX2 expression. After 21 days of induction by retinoic acid, expression of neural markers (neuroD1 and synaptophysin) was higher in induced cell clones than in induced parental cells. The cell clone with SOX2 overexpression had an approximately 1.3-fold higher growth rate compared to parental cells. SOX2 overexpression did not increase the population of cells undergoing apoptosis. Taken together, we developed two SOX2-overexpressing cell clones, with constitutive SOX2 expression after three weeks of retinoic acid treatment. SOX2 overexpression resulted in altered expression of pluripotency-related genes, increased proliferation, and altered expression of neural markers after three weeks of retinoic acid treatment.

First Report of Oidium neolycopersici on Greenhouse Tomatoes in Serbia.

In September 2011, tomato (Solanum lycopersicum L. 'Big Beef') plants showing typical symptoms of powdery mildew were collected in a greenhouse in the vicinity of Padinska Skela (District of City of Belgrade) in Serbia. Numerous circular, white colonies of powdery mildew were observed predominantly on the adaxial surface of the leaves, the petioles, and the stems. The foliage of infected plants turned yellow and necrotic, which was followed by rapid defoliation. Disease incidence was estimated by counting plants with powdery mildew symptoms in a random batch of 100 plants in four replicates and estimated to be extremely high, approaching 90%. Tomato plants ('Novosadski Jabucar') were inoculated with conidia released from diseased tomato leaves positioned above the tomato leaves and maintained at 25°C with a 14-h photoperiod. Healthy tomato plants from the same lot, which were not exposed to the conidia shower, were used as negative control. The first white fungal colonies appeared on the leaves of the inoculated plants within 4 to 7 days after inoculation, while no fungal growth was observed in the control plants. To determine the morphological characteristics of the pathogen, surface mycelium was removed with small strips of clear adhesive tape and examined using light microscopy. Microscopic observation revealed mycelium with lobed appressoria and hyaline, ellipsoid-ovoid or doliform conidia (32.5 to 47.5 × 17.5 to 25 µm) with no distinct firosin bodies and which produced sub-terminal germ tubes. Conidia were produced on the unbranched, erect conidiophores (82.5 to 150 µm) consisting of a cylindrical foot-cell followed by one to three short cells. No chasmothecia were found. On the basis of morphological characteristics, the pathogen was identified as Oidium neolycopersici (4), which was confirmed by internal transcribed spacer (ITS) sequence analysis. Total DNA was extracted directly from the whitish spots of superficial mycelium on the leaves with a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. PCR amplification and sequencing were performed with primers ITS1F and ITS4 (1). The nucleotide sequence of the representative isolate 809-11 (Accession No. JQ619840) shared 100% identity with 16 O. neolycopersici isolates deposited in GenBank from different parts of the world. Tomato powdery mildew caused by O. neolycopersici is present in many European (4) and other countries around the world (3) and is becoming economically very important as majority of the tomato cultivars have shown to be susceptible (2). To our knowledge, this is the first report of O. neolycopersici in Serbia. Because tomato is a very popular and widely grown vegetable in Serbia, the presence of a new and potentially harmful disease could endanger greenhouse as well as open field tomato production. References: (1) J. H. Cunnington et al. Australas. Plant Pathol. 32:421, 2003. (2) T. Jankovics et al. Phytopathology 98:529, 2008. (3) H. Jones et al. Mol. Plant Pathol. 2:303, 2001. (4) L. Kiss et al. Mycol. Res. 105:684, 2001.

Management of large peripheral nerve defects with autografting.

INTRODUCTION: A segmental nerve defect from trauma results in significant loss of function of the extremity, and rarely occurs in isolation. Autografting of the nerve defect is the current gold standard. METHODS: A review of the recent literature regarding peripheral nerve defects after trauma treated with autograft. RESULTS: Identification of the zone of nerve injury is difficult and appropriate resection is critical for good outcomes. Meaningful recovery is more likely with application of excellent technique. Many of the factors affecting outcomes are not modifiable. CONCLUSION: Nerve grafting for segmental nerve injuries continues to be an essential and appropriate treatment.

Morphological changes of poly(DI-lactide-co-glycolide) nano-particles containing ascorbic acid during in vitro degradation process.

Poly(DL-lactide-co-glycolide) powder composed of uniform particles with the mean particle size in the range of 110-170 nm was obtained from commercial granules. Ascorbic acid in different concentrations was encapsulated into the poly(DL-lactide-co-glycolide) particles. Degradation of the latter in terms of morphological changes in the physiological solution was followed. Within a period of 2 months, the particles completely degrade and all the ascorbic acid is released. The samples were characterized by ultraviolet spectroscopy and scanning electron microscopy.

Moisture and gamma-ray irradiation effects on the mechanical properties of carbon fibre-reinforced plastics.

The effects of gamma-irradiation and moisture absorption on the mechanical properties of carbon fibres-epoxy resin composites were studied. The properties dominated by the matrix and fibre-matrix interface (interlaminar and in-plane shear strength) were measured at room temperature using standard tests. These tests were carried out before and after exposures to gamma irradiation and before and after immersion in water at 80 degrees C during 21 days. The dosage of gamma irradiation was up to 11.7 MGy. The micrographs of surfaces fractured in performed tests were observed on a scanning electron microscope. They were analyzed with consulting the stated effects on mechanical properties and the measured values of the glass transition temperature of tested coupons before and after irradiation and immersion in water. The obtained results show that moisture and irradiation, if they act one after the other, have a significant influence on the degradation of matrix-dominated mechanical properties of the tested carbon-epoxy composite.

Complete division of the radial nerve associated with a closed fracture of the humeral shaft in a child.

The management of closed fractures of the humerus with an associated nerve palsy remains controversial. With very little written about this injury in children, we present the case of a three-year-old child with a closed humeral shaft fracture in whom surgical exploration and reconstruction of the radial nerve with a sural nerve graft was performed three months after injury. The child regained full function. To the best of our knowledge, this is the first such case to be reported in the English literature.

The human SOX18 gene: cDNA cloning and high resolution mapping.

SOX genes comprise a family of genes that are related to the mammalian sex determining gene SRY and these genes play key roles during animal development. We report here cloning and characterisation of the human SOX18 gene. SOX18 gene is expressed in foetal brain as well as in a wide range of foetal and adult tissues indicating its function is not restricted to early development. Mapping analysis has revealed that SOX18 gene is located on human chromosome 20q13.3, 27.29 cR distal from the marker D20S173.

Variant chromosomal arrangement of adult beta-globin genes in rat.

The genomic organization of three haplotypes of beta-globin genes was determined to resolve the question of the number of those genes in rat. Haplotype a, found in inbred strain DA, has three genes or pseudogenes, while haplotypes b, found in AO, Y5 and Wistar strains, and c, found in Wistar strain, have five genes or pseudogenes each. In haplotypes b and c, the first gene is of beta major type and the remaining four are of beta minor type. Partial sequencing of six out of 13 genes shows that duplications of beta minor genes are causing polymorphism in a number of genes. Also, in haplotype b two beta minor genes have a 6.5-kb intron 2, while in haplotype c only one beta minor gene contains such a large intron 2. The three structurally different haplotypes described are not interconvertible by single recombination events. The results indicate that the rat has the highest number of adult beta-globin genes found in mammals so far.

Incorporation of polylactide-polyglycolide in a cortical defect: neoangiogenesis and blood supply in a bone chamber.

Erodible polymers are an alternative to metals for fracture fixation (for example, in the malleolus) and for maxillofacial reconstruction. In this study, the vascular response to eroding polylactide-polyglycolide copolymer threads was observed chronically in a bone chamber implant, with use of intravital microscopy. A bone chamber implant loaded with 100 microns thick polylactide-polyglycolide threads was implanted into the right tibia in 15 mature female New Zealand White rabbits. Periodic intravital microscopic observations were performed from the third to the tenth or twelfth week after implantation. Vascularization, blood flow, and trabecular growth into the chambers from the medial cortex were recorded on videotape and analyzed using digital image processing. A statistically significant delay of neo-osteogenesis in the presence of this copolymer was described in an earlier report. The present report describes the measures of neoangiogenesis and blood supply; there was a significant delay in neoangiogenesis. It is suggested that both delayed angiogenesis and osteogenesis were secondary consequences of the macrophage response to slowly eroding poly-L-lactide crystal nanoparticles and the influence of reduced nutrient exchange. The lesser effect on blood supply and vascular volume fraction was seen to be linked to the slowing down of angiogenesis, as the latter allowed vessels to mature, with a widening of their calibers. This homeostatic adjustment was interpreted as being only partially successful in restoring control levels of oxygen delivery, because resulting increases in vessel surface area did not reach control levels. Thus, in the presence of eroding polylactide-polyglycolide, the oxygen supply and extravasation of other nutrients may be below normal during healing phases when the need is critical.

Tissue pressures in pyogenic flexor tenosynovitis of the finger. Compartment syndrome and its management.

We investigated 14 patients with pyogenic flexor tenosynovitis for increased tissue pressures in involved digits. All showed raised pressures, in eight to 30 mmHg or more. These levels are consistent with a compartment syndrome. We describe the results of a modified operative technique which includes irrigation of the sheath and the leaving open of a lateral incision. This also allows early active mobilisation of the finger and has given satisfactory early results.

Genotoxicity testing of cooked cured meat pigment (CCMP) and meat emulsion coagulates prepared with CCMP.

The preformed cooked cured meat pigment (CCMP) synthesized directly from bovine red blood cells or through a hemin intermediate was found to be a viable colorant for application to comminuted pork as a nitrite substitute. However the genotoxicity of CCMP and meat emulsion coagulates prepared with CCMP has not been evaluated. Therefore the objectives of this work were to investigate genotoxicity of CCMP and the influence of CCMP addition on genotoxicity and the content of residual nitrite in model meat emulsion coagulates. Meat emulsions were prepared from white (musculus longissimus dorsi) and red (musculus quadriceps femoris) pork muscles with two different amounts of synthesized pigment CCMP. Comparatively, emulsions with fixed addition of nitrite salt and emulsions without any addition for color development were made. Genotoxicity of CCMP and meat emulsion coagulates was tested with the SOS/umu test and the Ames test. Neither CCMP nor meat emulsion coagulates prepared with CCMP or nitrite salt were genotoxic in the SOS/umu test. In the Ames test using Salmonella Typhimurium strains TA98 and TA100 samples of coagulates prepared with CCMP and with nitrite showed weak mutagenic activity in Salmonella Typhimurium strain TA100 but only in the absence of the metabolic activation, while CCMP was not mutagenic. Coagulates prepared with CCMP contained significantly less residual nitrite than coagulates prepared with nitrite salt. These results indicate that from the human health standpoint the substitution of nitrite salt with CCMP would be highly recommendable.

Avulsion injuries of the thumb.

Avulsion amputations of the thumb are generally thought to have a worse prognosis after replantation than other amputations. We report the results of 17 thumbs that had an avulsion amputation and were replanted. Fourteen of the 17 survived (82 percent). Our experience indicated that the survival rate was improved by restoring continuity of at least two veins and two arteries, using a Y-shaped vein graft and the princeps pollicis artery for the source of arterial circulation. Nerve grafts were used to bridge defects in avulsed digital nerves. When possible, avulsed tendons were reattached to their muscle. Key pinch strength was 60 percent of normal, and grip strength was always less than that of the normal hand. The age of the patients and the cold ischemia time had no significant effect on either survival or function of the replanted thumb. When excellent venous backflow occurred immediately after the vessel repair and continued for at least 20 minutes, the thumb always survived without complications.

Simultaneous fractures of the distal end of the radius and the scaphoid bone.

Simultaneous fracture of the scaphoid bone occurred in 26 (4%) of the 650 injuries of the distal end of the radius seen at our institution. These injuries occur after a fall on the outstretched hand with a pronated wrist joint and extended hand. In a 4-year period (1983-1987), 26 simultaneous fractures of the distal end of the radius and the scaphoid bone were seen. Typically, the fracture of the radius had minimal or slight displacement, and the fracture of the scaphoid bone occurred always as a transverse thin line without displacement. The simultaneous fracture of the scaphoid bone was often very difficult to recognize radiographically and could very easily be overlooked. Discovery of the simultaneous fracture is important for adequate immobilization. Inadequate treatment, due to an overlooked fracture of the scaphoid bone, can result in a painful wrist joint and, possibly, Sudeck's atrophy.