Multidrug-Resistant Mycobacterium tuberculosis of the Latin American Mediterranean Lineage,Wrongly Identified as Mycobacterium pinnipedii (Spoligotype International Type 863 [SIT863]), Causing Active Tuberculosis in South Brazil

We recently detected the spoligotype patterns of strains of Mycobacterium pinnipedii, a species of the Mycobacterium tuberculosis complex, in sputum samples from nine cases with pulmonary tuberculosis residing in Porto Alegre, South Brazil. Because this species is rarely encountered in humans, we further characterized these nine isolates by additional genotyping techniques, including 24-locus mycobacterial interspersed repetitive-unit–variable-number tandem-repeat (MIRU-VNTR) typing, verification of the loci TbD1, RD9, pks15/1, RD^Rio, and fbpC, the insertion of IS6110 at a site specific to the M. tuberculosis Latin American Mediterranean (LAM) lineage, and whole-genome sequencing. The combined analysis of these markers revealed that the isolates are in fact M. tuberculosis and more specifically belong to the LAM genotype. Most of these isolates (n = 8) were shown to be multidrug resistant (MDR), which prompted us to perform partial sequencing of the rpoA, rpoB, rpoC, katG, and inhA genes. Seven isolates (77.8%) carried the S315T mutation in katG, and one of these (11%) also presented the C(−17)T single-nucleotide polymorphism (SNP) in inhA. Interestingly, six of the MDR isolates also presented an undescribed insertion of 12 nucleotides (CCA GAA CAA CCC) in codon 516 of rpoB. No putative compensatory mutation was found in either rpoA or rpoC. This is the first report of an M. tuberculosis LAM family strain with a convergent M. pinnipedii spoligotype. These spoligotypes are observed in genotype databases at a modest frequency, highlighting that care must be taken when identifying isolates in the M. tuberculosis complex on the basis of single genetic markers.     

Linear ubiquitination in immunity

Linear ubiquitination is a post-translational protein modification recently discovered to be crucial for innate and adaptive immune signaling. The function of linear ubiquitin chains is regulated at multiple levels: generation, recognition, and removal. These chains are generated by the linear ubiquitin chain assembly complex (LUBAC), the only known ubiquitin E3 capable of forming the linear ubiquitin linkage de novo. LUBAC is not only relevant for activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) in various signaling pathways, but importantly, it also regulates cell death downstream of immune receptors capable of inducing this response. Recognition of the linear ubiquitin linkage is specifically mediated by certain ubiquitin receptors, which is crucial for translation into the intended signaling outputs. LUBAC deficiency results in attenuated gene activation and increased cell death, causing pathologic conditions in both, mice, and humans. Removal of ubiquitin chains is mediated by deubiquitinases (DUBs). Two of them, OTULIN and CYLD, are constitutively associated with LUBAC. Here, we review the current knowledge on linear ubiquitination in immune signaling pathways and the biochemical mechanisms as to how linear polyubiquitin exerts its functions distinctly from those of other ubiquitin linkage types.     

ERIC-PCR genotyping of field isolates of Salmonella enterica subsp. enterica serovar Gallinarum biovars Gallinarum and Pullorum

Salmonella Gallinarum (SG) and Salmonella Pullorum (SP) have been classified as biovars belonging to Salmonella enterica subsp. enterica serovar Gallinarum. Genetic diversity among isolates of the same biovar can be detected by DNA fingerprinting techniques which are useful in epidemiological investigations. In this study, we applied the PCR amplification of Enterobacterial Repetitive Intergenic Consensus sequences (ERIC-PCR) to analyse 45 strains of SG and SP, most of which were isolated from diseased poultry of different Brazilian regions over a period of 27 years until 2014. The ERIC-genotypes obtained were used to describe the epidemiological relationship amongst the strains. Our findings showed that there were six ERIC-patterns for SG strains at 80% similarity. In addition, some of the SG isolates recovered from different regions and years clustered with 100% similarity, suggesting that transfer of genotypes between these regions has taken place. The commercial rough vaccine strain 9R showed a unique profile. Meanwhile, more genetic diversity was observed among SP strains where ten ERIC-patterns were also formed at 80% similarity.     

Narratives,memorable cases and metaphors of night nursing: findings from an interpretative phenomenological study

The aim of the study was to explore the experiences of night nurses. An interpretative phenomenological study was undertaken, and 35 nurses working in Italian medical, surgical and intensive care units were purposely recruited. Data were gathered in 2010 by semi‐structured interviews, collecting nurses' narratives, memorable cases and metaphors, aimed at summarising the essence of work as a nurse during the night. The experience of night nursing is based on four interconnected themes: (i) working in a state of alert, (ii) growing by expanding autonomy and responsibility, (iii) assuring sensitive surveillance and (iv) experiencing deep intimacy. Memorable episodes were polarised along (i) expected/unexpected events; (ii) positive/negative epilogues; and (iii) life/death issues. Many of the emergent metaphors described working during the night as being in the middle of a space where an apparent calm scene takes place, but unpredictable factors may suddenly change the order of events and the outcomes, creating chaos. Working during the night alerts nurses, who increase autonomy, expanding their role and assuming more responsibility with respect to that assumed during daily shifts. The nurses' clinical reasoning is based on data they carefully listen to, and on the meaning that nurses give time by time to different noises and silence. While in the past a sense of companionships was reported, a loneliness or a ‘neutral’ experience concerning the relationships with colleagues seems to prevail during night nursing. Working night shifts is a complex task, and specific training must be assured to students/novices.     

Use of miRNA Response Sequences to Block Off-target Replication and Increase the Safety of an Unattenuated,Glioblastoma-targeted Oncolytic HSV

Glioblastoma multiforme (GBM) is an aggressive brain cancer for which there is no effective treatment. Oncolytic HSV vectors (oHSVs) are attenuated lytic viruses that have shown promise in the treatment of human GBM models in animals, but their efficacy in early phase patient trials has been limited. Instead of attenuating the virus with mutations in virulence genes, we engineered four copies of the recognition sequence for miR-124 into the 3′UTR of the essential ICP4 gene to protect healthy tissue against lytic virus replication; miR-124 is expressed in neurons but not in glioblastoma cells. Following intracranial inoculation into nude mice, the miR-124-sensitive vector failed to replicate or show overt signs of pathogenesis. To address the concern that this safety feature may reduce oncolytic activity, we inserted the miR-124 response elements into an unattenuated, human receptor (EGFR/EGFRvIII)-specific HSV vector. We found that miR-124 sensitivity did not cause a loss of treatment efficiency in an orthotopic model of primary human GBM in nude mice. These results demonstrate that engineered miR-124 responsiveness can eliminate off-target replication by unattenuated oHSV without compromising oncolytic activity, thereby providing increased safety.     

High nevus counts confer a favorable prognosis in melanoma patients

A high number of nevi is the most significant phenotypic risk factor for melanoma and is in part genetically determined. The number of nevi decreases from middle age onward but this senescence can be delayed in patients with melanoma. We investigated the effects of nevus number count on sentinel node status and melanoma survival in a large cohort of melanoma cases. Out of 2,184 melanoma cases, 684 (31.3%) had a high nevus count (>50). High nevus counts were associated with favorable prognostic factors such as lower Breslow thickness, less ulceration and lower mitotic rate, despite adjustment for age. Nevus count was not predictive of sentinel node status. The crude 5‐ and 10‐year melanoma‐specific survival rate was higher in melanomas cases with a high nevus count compared to those with a low nevus count (91.2 vs. 86.4% and 87.2 vs. 79%, respectively). The difference in survival remained significant after adjusting for all known melanoma prognostic factors (hazard ratio [HR] = 0.43, confidence interval [CI] = 0.21–0.89). The favorable prognostic value of a high nevus count was also seen within the positive sentinel node subgroup of patients (HR = 0.22, CI = 0.08–0.60). High nevus count is associated with a better melanoma survival, even in the subgroup of patients with positive sentinel lymph node. This suggests a different biological behavior of melanoma tumors in patients with an excess of nevi.