High Dose Rate Brachytherapy for Inoperable Endometrial Cancer: a Case Series and Systematic Review of the Literature.

Endometrial cancer is a common gynaecological cancer, is typically early stage and treated with surgery. For patients where surgery is difficult or dangerous, definitive radiation therapy is the next best option. This study included a single institution case series (step 1) and a systematic review of the literature (step 2). In step 1, all endometrial cancer cases that were treated with definitive image-guided brachytherapy at a single institution from 2008 to 2020 were retrospectively analysed. In step 2, a systematic review of Medline (PubMed) from 1975 to 2020 was carried out using the key words around endometrial cancer and brachytherapy, followed by a narrative synthesis. In total, in step 1, 31 cases were included in this study, stages I-IV, with 96.7% receiving external beam radiation. All patients received three fractions of 7.5 Gy or five fractions of 6 Gy high dose rate brachytherapy, with a median EQD2 of 75.55 (40-84.3). The 2-year Kaplan-Meier (KM) local control was 83.1% and the 2-year KM overall survival was 77.4%. There was no late toxicity ≥grade 3. In step 2, 19 articles were included in the final analysis, with between six and 280 patients. The local control ranged from 70 to 100%, with low toxicity. Definitive radiation therapy with image-guided brachytherapy seems to have good local control with low toxicity for patients who are poor surgical candidates.

The Abl-1 Kinase is Dispensable for NK Cell Inhibitory Signalling and is not Involved in Murine NK Cell Education.

Natural killer (NK) cell responsiveness in the mouse is determined in an education process guided by inhibitory Ly49 and NKG2A receptors binding to MHC class I molecules. It has been proposed that inhibitory signalling in human NK cells involves Abl-1 (c-Abl)-mediated phosphorylation of Crk, lowering NK cell function via disruption of a signalling complex including C3G and c-Cbl, suggesting that NK cell education might involve c-Abl. Mice deficient in c-Abl expression specifically in murine NK cells displayed normal inhibitory and activating receptor repertoires. Furthermore, c-Abl-deficient NK cells fluxed Ca2+ normally after triggering of ITAM receptors, killed YAC-1 tumour cells efficiently and showed normal, or even slightly elevated, capacity to produce IFN-γ after activating receptor stimulation. Consistent with these results, c-Abl deficiency in NK cells did not affect NK cell inhibition via the receptors Ly49G2, Ly49A and NKG2A. We conclude that signalling downstream of murine inhibitory receptors does not involve c-Abl and that c-Abl plays no major role in NK cell education in the mouse.

MicroRNA-203 functions as a tumor suppressor in basal cell carcinoma.

Basal cell carcinoma (BCC) of the skin represents the most common malignancy in humans. MicroRNAs (miRNAs), small regulatory RNAs with pleiotropic function, are commonly misregulated in cancer. Here we identify miR-203, a miRNA abundantly and preferentially expressed in skin, to be downregulated in BCCs. We show that activation of the Hedgehog (HH) pathway, critically involved in the pathogenesis of BCCs, as well as the EGFR/MEK/ERK/c-JUN signaling pathway suppresses miR-203. We identify c-JUN, a key effector of the HH pathway, as a novel direct target for miR-203 in vivo. Further supporting the role of miR-203 as a tumor suppressor, in vivo delivery of miR-203 mimics in a BCC mouse model results in the reduction of tumor growth. Our results identify a regulatory circuit involving miR-203 and c-JUN, which provides functional control over basal cell proliferation and differentiation. We propose that miR-203 functions as a 'bona fide' tumor suppressor in BCC, whose suppressed expression contributes to oncogenic transformation via derepression of multiple stemness- and proliferation-related genes, and its overexpression could be of therapeutic value.

A fast, fully automated cell segmentation algorithm for high-throughput and high-content screening.

High-throughput, high-content screening (HT-HCS) of large compound libraries for drug discovery imposes new constraints on image analysis algorithms. Time and robustness are paramount while accuracy is intrinsically statistical. In this article, a fast and fully automated algorithm for cell segmentation is proposed. The algorithm is based on a strong attachment to the data that provide robustness and have been validated on the HT-HCS of large compound libraries and different biological assays. We present the algorithm and its performance, a description of its advantages and limitations, and a discussion of its range of application.

Balancing the role of the dental school in teaching, research and patient care; including care for underserved areas.

Inequalities within dentistry are common and are reflected in wide differences in the levels of oral health and the standard of care available both within and between countries and communities. Furthermore there are patients, particularly those with special treatment needs, who do not have the same access to dental services as the general public. The dental school should aim to recruit students from varied backgrounds into all areas covered by the oral healthcare team and to train students to treat the full spectrum of patients including those with special needs. It is essential, however, that the dental student achieves a high standard of clinical competence and this cannot be gained by treating only those patients with low expectations for care. Balancing these aspects of clinical education is difficult. Research is an important stimulus to better teaching and better clinical care. It is recognized that dental school staff should be active in research, teaching, clinical work and frequently administration. Maintaining a balance between the commitments to clinical care, teaching and research while also taking account of underserved areas in each of these categories is a difficult challenge but one that has to be met to a high degree in a successful, modern dental school.

Critical role of Qa1b in the protection of mature dendritic cells from NK cell-mediated killing.

Molecular interactions in natural killer (NK) cell-mediated killing of dendritic cells (DC) have under recent years come under scrutiny. Upon stimulation with IFN-gamma or lipopolysaccharide, DC become relatively resistant to NK cell-mediated lysis. In the present study, we investigated the role of Qa1(b) on DC and its receptor NKG2A on NK cells in the protection of mature DC from NK cells. We demonstrate that while both NKG2A+ and NKG2A- NK cells can efficiently lyse unstimulated DC, NKG2A+ NK cells but not NKG2A- NK cells are largely impaired in their ability to lyse mature DC. Similarly, mature DC from mice expressing H-2D(b), whose leader peptide sequence binds and stabilizes Qa1(b), were resistant to NK cell-mediated killing, suggesting that stable Qa1(b) expression contributes to the protection of mature DC. This finding was further validated by the demonstration that addition of the Qdm leader peptide could protect TAP1-/- DC from NK cell-mediated lysis both in vitro and in vivo. The present data suggest that stable expression of Qa1 on the surface of mature DC contributes to the protection of DC from NK cell-mediated lysis.

IFN-gamma production and degranulation are differentially regulated in response to stimulation in murine natural killer cells.

Activation of natural killer (NK) cells is induced via receptors like NKG2D, NKR-P1C and NKp46. This activation is balanced by interactions with inhibitory receptors. NK cell activation can lead to cytotoxicity mediated via polarized exocytosis of secretory lysosomes (degranulation) and interferon (IFN)-gamma production. We studied cell surface mobilization of a molecule present in secretory lysosomes, CD107a (LAMP-1), to monitor the relationship between degranulation of NK cells and their production of IFN-gamma at the single cell level. A comparison of responses in naive mouse NK cells and NK cells pre-activated with the type I interferon-inducer tilorone demonstrated a dramatic influence of pre-activation, allowing potent degranulation and IFN-gamma responses to NKG2D mediated stimulation that were not observed with naive NK cells. Degranulation and IFN-gamma production were performed by overlapping NK cell populations with generally higher frequencies of degranulating than IFN-gamma producing NK cells. An NK cell subset analysis based on expression of Mac-1 and CD27 revealed that immature NK cells (Mac-1(lo) CD27(hi)) are preferentially degranulating, Mac-1(hi) CD27(hi) cells perform both effector functions efficiently, while the most mature (Mac-1(hi) CD27(lo)) NK cells display reduced degranulation but with maintained IFN-gamma production.

Proteomic identification of M. tuberculosis protein kinase substrates: PknB recruits GarA, a FHA domain-containing protein, through activation loop-mediated interactions.

Genes for functional Ser/Thr protein kinases (STPKs) are ubiquitous in prokaryotic genomes, but little is known about their physiological substrates and their actual involvement in bacterial signal transduction pathways. We report here the identification of GarA (Rv1827), a Forkhead-associated (FHA) domain-containing protein, as a putative physiological substrate of PknB, an essential Ser/Thr protein kinase from Mycobacterium tuberculosis. Using a global proteomic approach, GarA was found to be the best detectable substrate of the PknB catalytic domain in non-denatured whole-cell protein extracts from M. tuberculosis and the saprophyte Mycobacterium smegmatis. Enzymological and binding studies of the recombinant proteins demonstrate that docking interactions between the activation loop of PknB and the C-terminal FHA domain of GarA are required to enable efficient phosphorylation at a single N-terminal threonine residue, Thr22, of the substrate. The predicted amino acid sequence of the garA gene, including both the N-terminal phosphorylation motif and the FHA domain, is strongly conserved in mycobacteria and other related actinomycetes, suggesting a functional role of GarA in putative STPK-mediated signal transduction pathways. The ensuing model of PknB-GarA interactions suggests a substrate recruitment mechanism that might apply to other mycobacterial kinases bearing multiple phosphorylation sites in their activation loops.

A new evolutionary scenario for the Mycobacterium tuberculosis complex.

The distribution of 20 variable regions resulting from insertion-deletion events in the genomes of the tubercle bacilli has been evaluated in a total of 100 strains of Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium canettii, Mycobacterium microti, and Mycobacterium bovis. This approach showed that the majority of these polymorphisms did not occur independently in the different strains of the M. tuberculosis complex but, rather, resulted from ancient, irreversible genetic events in common progenitor strains. Based on the presence or absence of an M. tuberculosis specific deletion (TbD1), M. tuberculosis strains can be divided into ancestral and "modern" strains, the latter comprising representatives of major epidemics like the Beijing, Haarlem, and African M. tuberculosis clusters. Furthermore, successive loss of DNA, reflected by region of difference 9 and other subsequent deletions, was identified for an evolutionary lineage represented by M. africanum, M. microti, and M. bovis that diverged from the progenitor of the present M. tuberculosis strains before TbD1 occurred. These findings contradict the often-presented hypothesis that M. tuberculosis, the etiological agent of human tuberculosis evolved from M. bovis, the agent of bovine disease. M. canettii and ancestral M. tuberculosis strains lack none of these deleted regions, and, therefore, seem to be direct descendants of tubercle bacilli that existed before the M. africanum-->M. bovis lineage separated from the M. tuberculosis lineage. This observation suggests that the common ancestor of the tubercle bacilli resembled M. tuberculosis or M. canettii and could well have been a human pathogen already.

6-oxocytidine containing oligonucleotides inhibit the HIV-1 integrase in vitro.

Integration of the proviral DNA into the genome of infected cells is a key step of HIV-1 replication. Integration is catalyzed by the viral enzyme integrase (IN). 6-oxocytidine-containing oligonucleotides were found to be efficient inhibitors of integrase in vitro. The inhibitory effect is sequence-specific and strictly requires the presence of the 6-oxocytidine base. It is due to the impairment of the integrase binding to its substrate and does not involve an auto-structure of the oligonucleotide.

The acute effects of smokeless tobacco (snuff) on gingival blood flow in man.

Snuff-induced blood flow responses in the gingiva were evaluated in 22 healthy casual consumers of tobacco. Laser Doppler flowmetry (LDF) was used to measure blood flow simultaneously and continuously on two gingival sites (buccal aspect of the papillae between the upper lateral incisors and canines). In addition, measurements of skin blood flow in the forehead and palmar side of the left thumb were performed. Arterial blood pressure (BP) and heart rate (HR) were also recorded. Unilateral application of commercial snuff (500 mg, 1%) caused a marked and rapid increase in gingival blood flow (GBF) on the exposed side (p < 0.001). Blood flow increased also in the contralateral gingiva and forehead skin (p < 0.05). Skin blood flow in the thumb showed an insignificant decrease. BP and HR increased. Vascular conductance increased significantly in the snuff-exposed gingiva but not in the contralateral gingiva or the forehead. Vascular conductance was largely unaffected in the thumb. It is concluded that acute application of snuff, besides giving rise to typical changes in BP and HR, increases GBF in and around the exposed area, probably through activation of sensory nerves and the subsequent release of vasodilatory peptides from their peripheral endings. Blood flow in unexposed gingival and forehead skin may increase probably due to humoral or nervously mediated mechanisms. However, a passive pressure-induced hyperaemia in the unexposed gingiva and forehead skin can not be excluded.

Determinants of Mg2+-dependent activities of recombinant human immunodeficiency virus type 1 integrase.

The relationship between Mg(2+)-dependent activity and the self-assembly state of HIV-1 integrase was investigated using different protein preparations. The first preparations, IN(CHAPS) and IN(dial), were purified in the presence of detergent, but in the case of IN(dial), the detergent was removed during a final dialysis. The third preparation, IN(zn), was purified without any detergent. The three preparations displayed comparable Mn(2+)-dependent activities. In contrast, the Mg(2+)-dependent activity that reflects a more realistic view of the physiological activity strongly depended on the preparation. IN(CHAPS) was not capable of using Mg(2+) as a cofactor, whereas IN(zn) was highly active under the same conditions. In the accompanying paper [Deprez, E., et al. (2000) Biochemistry 39, 9275-9284], we used time-resolved fluorescence anisotropy to demonstrate that IN(CHAPS) was monomeric at the concentration of enzymatic assays. Here, we show that IN(zn) was homogeneously tetrameric under similar conditions. Moreover, IN(dial) that exhibited an intermediary Mg(2+)-dependent activity existed in a monomer-multimer equilibrium. The level of Mg(2+)- but not Mn(2+)-dependent activity of IN(dial) was altered by addition of detergent which plays a detrimental role in the maintenance of the oligomeric organization. Our results indicate that the ability of integrase to use Mg(2+) as a cofactor is related to its self-assembly state in solution, whereas Mn(2+)-dependent activity is not. Finally, the oligomeric IN(zn) was capable of binding efficiently to DNA regardless of the cationic cofactor, whereas the monomeric IN(CHAPS) strictly required Mn(2+). Thus, we propose that a specific conformation of integrase is a prerequisite for its binding to DNA in the presence of Mg(2+).

Branched oligonucleotide-intercalator conjugate forming a parallel stranded structure inhibits HIV-1 integrase.

Integration of a DNA copy of the HIV-1 genome into chromosomal DNA of infected cells is a key step of viral replication. Integration is carried out by integrase, a viral protein which binds to both ends of viral DNA and catalyses reactions of the 3'-end processing and strand transfer. A 3'-3' branched oligonucleotide functionalised by the intercalator oxazolopyridocarbazole at each 5'-end was found to inhibit integration in vitro. We show that both a specific (G,A) sequence and the OPC intercalating agent contribute to the capability of the branched oligonucleotide to form a parallel stranded structure responsible for the inhibition.

Inhibition of HIV-1 integration by mono- & bi-functionalized triple helix forming oligonucleotides.

HIV-1 DNA integration is carried out by integrase, a viral protein which binds to specific sequences located on both extremities of the HIV-1 DNA LTR. Inhibition of integration was observed with submicromolar concentrations of mono- or bifunctionalized 11-mer oligonucleotide-intercalators, which were designed to form an alternate strand triple helix with the U5 LTR end containing two adjacent purine tracts on opposite strands 5'-GGAAAATCTCT-3'/3'-CCTTTTAGAGA-5'.

Optimization of alternate-strand triple helix formation at the 5"-TpA-3" and 5"-ApT-3" junctions.

Alternate-strand triple helix formation was optimized at the two junction steps, the 5"-TpA-3" and 5"-ApT-3" junctions. Footprint experiments, gel retardation assays and thermal denaturation measures on a sequence appropriately designed with two adjacent alternate-strand polypurine tracts points out that the addition of an adenine residue and the removal of one nucleotide should facilitate the crossing strands at the 5"-TpA-3" junction and at the 5"-ApT-3" junction, respectively. These results provide a 'switch code' for the construction of alternate-strand triple helix forming oligonucleotides which open new possibilities for extending the range of applications of antigene strategy.

NF-kappaB only partially mediates Epstein-Barr virus latent membrane protein 1 activation of B cells.

The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is required for EBV-induced immortalization of human B cells and causes tumorigenic transformation of cell lines. LMP1 expression induces phenotypic changes resembling B cell activation, such as cell size increase and up-regulation of cell surface activation markers. LMP1 contains two domains that activate the transcription factor NF-kappaB, one through interactions with TRAF proteins and the other with the TRADD protein. The purpose of the present study was to investigate the importance of NF-kappaB induction in the up-regulation of the B cell activation markers ICAM-1 and CD71 by LMP1. This study shows that expression of LMP1 activates transcription from p50/p65- and c-Rel-responsive promoters, and that this activity can be completely inhibited by expression of a dominant inhibitory IkappaB mutant. ICAM-1 and CD71 are nevertheless up-regulated by LMP1 in primary B cells and cell lines expressing the dominant IkappaB. Furthermore, LMP1-induced cell size increase of primary B cells was unaffected by IkappaB expression. It was concluded that even when LMP1 is unable to activate NF-kappaB, it is still capable of inducing certain characteristics of activated B cells, strongly suggesting that LMP1 can also activate cells independently of NF-kappaB.

Salivary secretion: stimulatory effects of chewing-gum versus paraffin tablets.

Paraffin tablets are commonly used in clinical saliva tests whereas chewing-gum is recommended to increase salivation in xerostomic patients. The aim of this study was to compare the effect on salivation of these stimuli. Saliva stimulated by chewing-gum or paraffin tablets was sampled on separate occasions from eight healthy subjects (25-45 yr). Whole or parotid saliva were collected for 6 min (1 min + 5 min) and 21 min (1 min + 5 min x 4), respectively. pH of saliva was measured with and without the addition of HC1 (titrated pH). Total parotid protein was measured using the microBSA-assay. Initially, flow rates were significantly higher during the chewing of gum vs. paraffin tablets (whole saliva 5.18 vs. 2.99 ml/min, parotid saliva 0.83 vs. 0.42 ml/min). Conversely, during final chewing periods, parotid flow rates, pH, and titrated pH were significantly higher during paraffin chewing. When comparing the stimuli, parotid protein output was higher initially during gum chewing, but in the final period paraffin chewing elicited the higher output. It was concluded that the clinical test of paraffin chewing gives a good estimate of the expected whole saliva response to chewing-gum. Furthermore, extended chewing of paraffin tablets seemed to influence parameters of parotid saliva positively.

Upregulation of growth associated protein 43 expression and neuronal co-expression with neuropeptide Y following inferior alveolar nerve axotomy in the rat.

Growth associated protein 43 (GAP 43) is an acidic membrane-bound phosphoprotein produced at high levels in developing and regenerating neurons. It is a substrate for protein kinase C and suggested to be involved in calcium-regulated release of axonal vesicular-contained neurotransmitters. Expression of GAP 43 has been demonstrated in the uninjured cat dental pulp which receives its sensory nerve supply from the trigeminal ganglion. The aim of this study was a detailed mapping of the spatial and time-dependent expression of GAP 43 and co-expression of neuropeptide Y (NPY) in dental peripheral target tissues and trigeminal neurons subsequent to inferior alveolar nerve (IAN) axotomy in rats, as background for later low-level laser studies. Unilateral sectioning of IAN, resulting in an almost complete loss of sensory nerve fibers in the ipsilateral dental pulp of the first molar, was performed. The avidin biotin complex (ABC) method was used to evaluate peripheral changes in GAP 43 expression at 4, 7 and 10 days. Ganglionic changes in GAP 43 and co-localization of neuronal NPY expression was examined at 4, 10 and 21 days using either the ABC method or double immunofluorescence labelling techniques and confocal microscopy. Axotomy resulted in an early upregulation and change in the peripheral distribution of GAP 43 in nerve profiles already 4 days post IAN axotomy suggesting a Schwann cell origin. Ten days post axotomy a pronounced upregulation of GAP 43 immunoreactivity could be demonstrated in neurons located in the mandibular region of the trigeminal ganglion, compared to the contralateral uninjured side. The peripheral and ganglionic upregulation of GAP 43 continued to persist at 21 days. A concomitant time-delayed shift and co-expression of NPY was demonstrated throughout in the GAP 43-upregulated ganglion cells 10 days post axotomy. Furthermore, confocal microscopy indicated that the intraneuronal distribution of NPY and upregulated GAP 43 expression showed a similar conformity and distribution in both perinuclear regions and cell periphery.

A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus.

Tat is an 86-amino acid protein involved in the replication of human immunodeficiency virus type 1 (HIV-1). Several studies have shown that exogenous Tat protein was able to translocate through the plasma membrane and to reach the nucleus to transactivate the viral genome. A region of the Tat protein centered on a cluster of basic amino acids has been assigned to this translocation activity. Recent data have demonstrated that chemical coupling of a Tat-derived peptide (extending from residues 37 to 72) to several proteins allowed their functional internalization into several cell lines or tissues. A part of this same domain can be folded in an alpha-helix structure with amphipathic characteristics. Such helical structures have been considered as key determinants for the uptake of several enveloped viruses by fusion or endocytosis. In the present study, we have delineated the main determinants required for Tat translocation within this sequence by synthesizing several peptides covering the Tat domain from residues 37 to 60. Unexpectedly, the domain extending from amino acid 37 to 47, which corresponds to the alpha-helix structure, is not required for cellular uptake and for nuclear translocation. Peptide internalization was assessed by direct labeling with fluorescein or by indirect immunofluorescence using a monoclonal antibody directed against the Tat basic cluster. Both approaches established that all peptides containing the basic domain are taken up by cells within less than 5 min at concentrations as low as 100 nM. In contrast, a peptide with a full alpha-helix but with a truncated basic amino acid cluster is not taken up by cells. The internalization process does not involve an endocytic pathway, as no inhibition of the uptake was observed at 4 degrees C. Similar observations have been reported for a basic amino acid-rich peptide derived from the Antennapedia homeodomain (1). Short peptides allowing efficient translocation through the plasma membrane could be useful vectors for the intracellular delivery of various non-permeant drugs including antisense oligonucleotides and peptides of pharmacological interest.