Bilateral unusual branching pattern of the external carotid artery in a human cadaver.

Comprehensive understanding of the variations in the branching of the external carotid artery (ECA) is essential to minimizing vascular complications during cranio-facial and neck surgical procedures. We demonstrate a rare case of unusual branching of ECAs in both carotid triangles and anomalous origin of the left ascending pharyngeal artery (APA) during dissection of embalmed cadaver. The right and left common carotid arteries (CCA) bifurcated at the level of the upper border of the thyroid cartilage. The right superior thyroid artery (STA) originated anterior to the carotid bifurcation (CB), while the left STA originated from the anterior aspect of the left CCA. The right ECA trifurcated into linguofacial trunk, APA, and distal ECA, 15.7 mm from CB. On the left side, lingual artery and APA arose as a short common linguopharyngeal trunk, 1.9 mm from CB. The left facial and occipital arteries originated anteromedially and posteriorly at the same level.

Risk factors for gut colonization with vancomycin-resistant enterococci among Bulgarian critically ill patients.

Vancomycin-resistant enterococci (VRЕ) are recognized as important hospital pathogens which have become common in patients admitted to the intensive care units (ICUs). The purpose of this study was to evaluate the incidence of and the risk factors for colonization with VRE among ICU patients. A total of 91 patients who had duration of hospitalization more than 48 h and without infection caused by VRE or/and other microorganisms in the ICU at University Hospital, Pleven were screened for colonization with VRE. The following data were collected: demographic characteristics, clinical information and antimicrobials use. The statistical analysis was performed using SPSS version 27.0. Colonization with VRE was established in 22 patients and one was carrying two enterococcal species. A total of 23 VRE were isolated. The univariate analysis showed that the postoperative critical cares (p < 0.001), cardiovascular diseases (p = 0.009) and the presence of an endotracheal tube (p = 0.003) were risk factors for colonization with VRE. Also, the postoperative critical cares (p = 0.021) and cardiovascular diseases (p = 0.018) were confirmed as independent risk factor for VRE acquisition by multivariate analysis. The prevalence of VRE colonization among the ICU patients was relatively high (24.2%). Risk factors for acquisition of intestinal VRE were the postoperative cares, cardiovascular diseases and the presence of an endotracheal tube.

Gut colonization with vancomicyn-resistant enterococci among patients with hematologic malignancies.

BACKGROUND: Vancomycin-resistant enterococci (VRE) are well known agents that colonize the gastrointestinal tract of immunocompromised patients, especially those with hematologic malignancies. The aim of the current study was to determine the incidence of and risk factors for colonization with VRE among patients with hematologic malignancies. MATERIALS: For a nine-month period, all patients admitted to the Hematology ward at University Hospital in Pleven, Bulgaria who had hematologic malignancy and duration of hospitalization of more than 48 h were screened for colonization with VRE. The data collected from patients and their medical records during the entire hospital stay included: demographic characteristics, clinical information and information about all antimicrobials used. A longitudinal study was used to assesses the risk factors and statistical analysis was performed using SPSS version 27.0. RESULTS: A total of 119 patients were enrolled in the study. Colonization with VRE was established in 18 of them. One patient carried two species, resulting in a total of 19 VRE: 12 Enterococcus gallinarum, 4 Enterococcus casseliflavus, 2 Enterococcus faecium and 1 Enterococcus faecalis. VanA phenotype, with high-level resistance of vancomycin (MIC ≥ 256 µg/ml) and teicoplanin (MIC = 96 µg/ml), was demonstrated by one E. faecium, which carried vanA. The other E. faecium and E. faecalis expressed low-level resistance to vancomycin (MICs: 8 µg/ml and 12 µg/ml), susceptibility to teicoplanin (MICs = 0.5 µg/ml) and vanB was detected. All E. gallinarum and E. casseliflavus showed low-level resistance to vancomycin and susceptibility to teicoplanin. E. gallinarum strains were positive for vanC1 and E. casseliflavus for vanC2. Only two patients were colonized with vanA or vanB enterococci and the rest 16 were positive for vanC. The univariate analysis revealed that patient's age (70-79 years; p = 0.025) and multiple myeloma (p = 0.001) are risk factors for VRE acquisition among the investigated patients. In addition, the multivariate analysis confirmed that patient's age (70-79 years) is an independent risk factor for VRE colonization. CONCLUSIONS: Our results showed that 15.1% of patients with hematologic malignancies were colonized with VRE. There was a distinct prevalence of vanC enterococci. Among the analyzed risk factors, advanced age and multiple myeloma contributed to VRE acquisition.

Overview and assessment of the histochemical methods and reagents for the detection of ß-galactosidase activity in transgenic animals.

Bacterial ß-galactosidase is one of the most widely used reporter genes in experiments involving transgenic and knockout animals. In this review we discuss the current histochemical methods and available reagents to detect ß-galactosidase activity. Different substrates are available, but the most commonly used is X-gal in combination with potassium ferri- and ferro-cyanide. The reaction produces a characteristic blue precipitate in the cells expressing ß-galactosidase, and despite its efficiency in staining whole embryos, its detection on thin tissue sections is difficult. Salmon-gal is another substrate, which in combination with ferric and ferrous ions gives a reddish-pink precipitate. Its sensitivity for staining tissue sections is similar to that of X-gal. Combining X-gal or Salmon-gal with tetrazolium salts provides a faster and more sensitive reaction than traditional ß-galactosidase histochemistry. Here, we compare the traditional ß-galactosidase assay and the combination of X-gal or Salmon-gal with three tetrazolium salts: nitroblue tetrazolium, tetranitroblue tetrazolium and iodonitrotetrazolium. Based on an assessment of the sensitivity and specificity of the different combinations of substrates, we are proposing an optimized and enhanced method for ß-galactosidase detection in histological sections of the transgenic mouse brain. Optimal staining was obtained with X-gal in combination with nitroblue tetrazolium, which provides a faster and more specific staining than the traditional X-gal combination with potassium ferri- and ferro-cyanide. We recommend the X-gal/nitroblue tetrazolium staining mixture as the first choice for the detection of ß-galactosidase activity on histological sections. When faster results are needed, Salmon-gal/nitroblue tetrazolium should be considered as an alternative, while maintaining acceptable levels of noise.

Glutamic acid decarboxylase 1 alternative splicing isoforms: characterization, expression and quantification in the mouse brain.

BACKGROUND: GABA has important functions in brain plasticity related processes like memory, learning, locomotion and during the development of the nervous system. It is synthesized by the glutamic acid decarboxylase (GAD). There are two isoforms of GAD, GAD1 and GAD2, which are encoded by different genes. During embryonic development the transcription of GAD1 mRNA is regulated by alternative splicing and several alternative transcripts were distinguished in human, mouse and rat. Despite the fact that the structure of GAD1 gene has been extensively studied, knowledge of its exact structural organization, alternative promoter usage and splicing have remained incomplete. RESULTS: In the present study we report the identification and characterization of novel GAD1 splicing isoforms (GenBank: KM102984, KM102985) by analyzing genomic and mRNA sequence data using bioinformatics, cloning and sequencing. Ten mRNA isoforms are generated from GAD1 gene locus by the combined actions of utilizing different promoters and alternative splicing of the coding exons. Using RT-PCR we found that GAD1 isoforms share similar pattern of expression in different mouse tissues and are expressed early during development. Quantitative RT-PCR was used to investigate the expression of GAD1 isoforms and GAD2 in olfactory bulb, cortex, medial and lateral striatum, hippocampus and cerebellum of adult mouse. Olfactory bulb showed the highest expression of GAD1 transcripts. Isoforms 1/2 are the most abundant forms. Their expression is significantly higher in the lateral compared to the medial striatum. Isoforms 3/4, 5/6, 7/8 and 9/10 are barely detectable in all investigated regions except of the high expression in olfactory bulb. When comparing GAD1 expression with GAD2 we found that Isoforms 1/2 are the predominant isoforms. In situ hybridization confirmed the predominant expression of Isoforms 7/8 and 9/10 in the olfactory bulb and revealed their weak expression in hippocampus, cerebellum and some other areas known to express GAD1. CONCLUSIONS: Generation of ten splicing isoforms of GAD1 was described including two so far uncharacterized transcripts. GAD1 splicing isoforms producing the shorter, enzymatically inactive GAD25 protein are expressed at very low level in adult mouse brain except in the olfactory bulb that is associated with neurogenesis and synaptic plasticity even during adulthood.

Lineage-specific purification of neural stem/progenitor cells from differentiated mouse induced pluripotent stem cells.

Since induced pluripotent stem (iPS) cells have differentiation potential into all three germ layer-derived tissues, efficient purification of target cells is required in many fields of iPS research. One useful strategy is isolation of desired cells from differentiated iPS cells by lineage-specific expression of a drug-resistance gene, followed by drug selection. With this strategy, we purified neural stem/progenitor cells (NSCs), a good candidate source for regenerative therapy, from differentiated mouse iPS cells. We constructed a bicistronic expression vector simultaneously expressing blasticidin S resistance gene and DsRed under the control of tandem enhancer of a 257-base pair region of nestin second intron, an NSC-specific enhancer. This construct was efficiently inserted into the iPS genome by piggyBac transposon-mediated gene transfer, and the established subclone was differentiated into NSCs in the presence or absence of blasticidin S. Consequently, incubation with blasticidin S led to purification of NSCs from differentiated iPS cells. Our results suggest that a lineage-specific drug selection strategy is useful for purification of NSCs from differentiated iPS cells and that this strategy can be applied for the purification of other cell types.

Lateral regions of the rodent striatum reveal elevated glutamate decarboxylase 1 mRNA expression in medium-sized projection neurons.

The GABA-synthesizing enzymes glutamate decarboxylase (GAD)1 and GAD2 are universally contained in GABAergic neurons in the central nervous system of the mouse and rat. The two isoforms are almost identically expressed throughout the brain and spinal cord. By using in situ hybridization, we found that the mouse lateral striatum concentrates medium-sized projection neurons with high-level expression of GAD1, but not of GAD2, mRNA. This was confirmed with several types of riboprobe, including those directed to the 5'-noncoding, 3'-noncoding and coding regions. Immunohistochemical localization of GAD1 also revealed predominant localization of the enzyme in the same striatal region. The lateral region of the mouse striatum, harboring such neurons, is ovoid in shape and extends between interaural +4.8 and +2.8, and at lateral 2.8 and dorsoventral 2.0. This intriguing region corresponds to the area that receives afferent inputs from the primary motor and sensory cortex that are presumably related to mouth and forelimb representations. The lateral striatum is included in the basal ganglia-thalamocortical loop, and is most vulnerable to various noxious stimuli in the neurodegeneration processes involving the basal ganglia. We have confirmed elevated expression of GAD1 mRNA, but not of GAD2 mRNA, also in the rat lateral striatum. Image analysis favored the view that the regional increase is caused by elevated cellular expression, and that the greatest number of medium-sized spiny neurons were positive for GAD1 mRNA. The GAD1 mRNA distribution in the mouse lateral striatum partially resembled those of GPR155 and cannabinoid receptor type 1 mRNAs, suggesting functional cooperation in some neurons.

Histological determination of the areas enriched in cholinergic terminals and M2 and M3 muscarinic receptors in the mouse central auditory system.

Cholinergic projections to auditory system are vital for coupling arousal with sound processing. Systematic search with in situ hybridization and immunohistochemistry indicated that the ventral nucleus of the medial geniculate body and the nucleus of the brachium of the inferior colliculus constituted cholinergic synaptic sites in the brainstem auditory system, containing a significant number of cholinergic axon terminals and m2 receptor-expressing cell bodies.

GPR155: Gene organization, multiple mRNA splice variants and expression in mouse central nervous system.

Emerging evidence suggests that GPR155, an integral membrane protein related to G-protein coupled receptors, has specific roles in Huntington disease and autism spectrum disorders. This study reports the structural organization of mouse GPR155 gene and the generation of five variants (Variants 1-5) of GPR155 mRNA, including so far unknown four variants. Further, it presents the level of expression of GPR155 mRNA in different mouse tissues. The mRNAs for GPR155 are widely expressed in adult mouse tissues and during development. In situ hybridization was used to determine the distribution of GPR155 in mouse brain. The GPR155 mRNAs are widely distributed in forebrain regions and have more restricted distribution in the midbrain and hindbrain regions. The highest level of expression was in the lateral part of striatum and hippocampus. The expression pattern of GPR155 mRNAs in mouse striatum was very similar to that of cannabinoid receptor type 1. The predicted protein secondary structure indicated that GPR155 is a 17-TM protein, and Variant 1 and Variant 5 proteins have an intracellular, conserved DEP domain near the C-terminal.