Phenotypic and genomic characterization of a small colony variant of Klebsiella pneumoniae isolated from urine of a prostate cancer case.

Small colony variants (SCVs) in Klebsiella pneumoniae are rare and understudied. We report an SCV of Klebsiella pneumoniae isolated from the urine of a prostate cancer patient undergoing prolonged radiotherapy. The strain was non-lactose fermenting, non-mucoid, slow-growing, multi-drug resistant, and showed atypical biochemical reactions and biofilm formation. On whole genome sequencing, it showed low-level virulence, sequence type 231 and gene CTX-M-15. Three major porins OmpK35, OmpK36 and OmpK37 were found. SCVs pose challenges like difficulties in identification, altered metabolism, and increased biofilm formation, which contribute to persistent infections. Radiotherapy and chemotherapy may have led to the formation of the SCV phenotype.

The WMI Rat of Premature Cognitive Aging Presents Intrinsic Vulnerability to Oxidative Stress in Primary Neurons and Astrocytes Compared to Its Nearly Isogenic WLI Control.

The primary neuronal and astrocyte culture described here is from the stress-hyperreactive Wistar Kyoto (WKY) More Immobile (WMI) rat with premature aging-related memory deficit, and its nearly isogenic control, the Less Immobile (WLI) strain. Primary WMI hippocampal neurons and cortical astrocytes are significantly more sensitive to oxidative stress (OS) generated by administration of H2O2 compared to WLI cells as measured by the trypan blue cell viability assay. Intrinsic genetic vulnerability is also suggested by the decreased gene expression in WMI neurons of catalase (Cat), and in WMI cortical astrocytes of insulin-like growth factor 2 (Igf2), synuclein gamma (Sncg) and glutathione peroxidase 2 (Gpx2) compared to WLI. The expressions of several mitochondrial genes are dramatically increased in response to H2O2 treatment in WLI, but not in WMI cortical astrocytes. We propose that the vulnerability of WMI neurons to OS is due to the genetic differences between the WLI and WMI. Furthermore, the upregulation of mitochondrial genes may be a compensatory response to the generation of free radicals by OS in the WLIs, and this mechanism is disturbed in the WMIs. Thus, this pilot study suggests intrinsic vulnerabilities in the WMI hippocampal neurons and cortical astrocytes, and affirm the efficacy of this bimodal in vitro screening system for finding novel drug targets to prevent oxidative damage in illnesses.

Salmonella enterica serovar Give causing brain abscess and bacteremia: A case report.

Non-typhoidal Salmonellosis are an important cause of gastroenteritis and invasive disease in developing countries, with increase resistance and mortality in paediatric age group. We report here, a rare case of bacteremia and brain abscess in a 3year old female child with Salmonella enterica serovar Give as a causative organism.

In Vitro and In Vivo Approaches to Study Kinetochore-Microtubule Attachments During Mitosis.

The separation of duplicated chromosomes during mitosis is a pivotal step in the process of cellular division. Therefore, the orchestrated events that take place to ensure proper attachment and stabilization of kMTs are keen areas of interest in the mitosis field. Here we describe the methods used to study kMT attachments via in vitro biochemical methods and in vivo cell biological approaches.

The formation of small aggregates contributes to the neurotoxic effects of tau45-230.

Intracellular deposits of hyperphosphorylated tau are commonly detected in tauopathies. Furthermore, these aggregates seem to play an important role in the pathobiology of these diseases. In the present study, we determined whether the recently identified neurotoxic tau45-230 fragment also formed aggregates in neurodegenerative disorders. The presence of such aggregates was examined in brain samples obtained from Alzheimer's disease (AD) subjects by means of Western blot analysis performed under non-denaturing conditions. Our results showed that a mixture of tau45-230 oligomers of different sizes was easily detectable in brain samples obtained from AD subjects. Our data also suggested that tau45-230 oligomers could be internalized by cultured hippocampal neurons, mainly through a clathrin-mediated mechanism, triggering their degeneration. In addition, in vitro aggregation studies showed that tau45-230 modulated full-length tau aggregation thereby inducing the formation of smaller, and potentially more toxic, aggregates of this microtubule-associated protein. Together, these data identified alternative mechanisms underlying the toxic effects of tau45-230.

Altered Cytoskeletal Composition and Delayed Neurite Elongation in tau45-230-Expressing Hippocampal Neurons.

Calpain-mediated tau cleavage into the neurotoxic tau45-230 fragment plays an important role in Alzheimer's disease (AD). This tau fragment accumulates mainly in the cytoplasm of degenerating neurons. However, subcellular localization studies indicated that a pool of tau45-230 associates with the cytoskeleton in hippocampal neurons. In the present study, we assessed whether such localization could underlie tau45-230 neurotoxic effects. Quantitative Western blot analysis showed decreased levels of full-length tau bound to microtubules in tau45-230-expressing hippocampal neurons when compared to controls. In addition, the presence of this tau fragment induced a transient increase in tyrosinated tubulin, a marker of unstable microtubules, followed by a significant decrease in the levels of this tubulin isoform. The data obtained also showed a significant reduction in actin filaments in tau45-230-expressing neurons. These changes in microtubules and actin filaments correlated with delayed neurite elongation and axonal differentiation in the presence of this tau fragment. Together, these results suggest that tau45-230 could exert its toxic effects, at least in part, by modifying the composition of the neuronal cytoskeleton and impairing neurite elongation in neurons undergoing degeneration.

Methods related to studying tau fragmentation.

Several tau posttranslational modifications have been implicated in neuronal degeneration. Among them, tau fragmentation has been identified not only in brain samples obtained from Alzheimer's disease (AD) and related disorder subjects but also in AD culture and animal models. Some of these tau fragments have not been extensively studied. In contrast, data obtained recently showed that tau fragmentation mediated by enhanced or abnormal calpain, caspase 2, caspase 3, and asparagine endopeptidase activity results in the formation of toxic fragments. These cleaved tau forms induce neuronal death, synapse loss, and/or behavioral deficits. Here, we described protease activity assays and methods to study the effects of tau fragments on neuronal viability.

Tau45-230 association with the cytoskeleton and membrane-bound organelles: Functional implications in neurodegeneration.

The dysregulation of posttranslational modifications of the microtubule-associated protein (MAP) tau plays a key role in Alzheimer's disease (AD) and related disorders. Thus, we have previously shown that beta amyloid (Aß)-induced neurotoxicity was mediated, at least in part, by tau cleavage into the tau45-230 fragment. However, the mechanisms underlying the toxicity of tau45-230 remain unknown. To get insights into such mechanisms, we first determined the subcellular localization of this tau fragment in hippocampal neurons. Tau45-230 was easily detectable in cell bodies and processes extended by these neurons. In addition, cell extraction experiments performed using Triton X-100 and saponin showed that a pool of tau45-230 was associated with the cytoskeleton and the cytoskeleton plus membrane-bound organelles, respectively, in cultured hippocampal neurons. Furthermore, they suggested that these associations were independent of the presence of full-length tau. We also assessed whether this tau fragment could alter axonal transport. Our results indicated that tau45-230 significantly reduced the number of organelles transported along hippocampal axons. This altered axonal transport did not correlate with changes in the total number of organelles present in these cells or in motor protein levels. Together these results suggested that tau45-230 could exert its toxic effects by partially blocking axonal transport along microtubules thus contributing to the early pathology of AD.

The Neurotoxic TAU45-230 Fragment Accumulates in Upper and Lower Motor Neurons in Amyotrophic Lateral Sclerosis Subjects.

Amyotrophic lateral sclerosis (ALS) is a progressive and lethal neurodegenerative disease characterized by the loss of upper and lower motor neurons leading to muscle paralysis in affected individuals. Numerous mechanisms have been implicated in the death of these neurons. However, the pathobiology of this disease has not been completely elucidated. In the present study, we investigated to what extent tau cleavage and the generation of the neurotoxic tau45-230 fragment is associated with ALS. Quantitative Western blot analysis indicated that high levels of tau45-230 accumulated in lumbar and cervical spinal cord specimens obtained from ALS subjects. This neurotoxic tau fragment was also detected in ALS upper motor neurons located in the precentral gyrus. Our results also showed that tau45-230 aggregates were present in the spinal cord of ALS patients. On the other hand, this neurotoxic fragment was not generated in a mouse model of a familial form of this disease. Together, these results suggest a potential role for this neurotoxic tau fragment in the mechanisms leading to the degeneration of motor neurons in the context of sporadic ALS.

The RZZ complex requires the N-terminus of KNL1 to mediate optimal Mad1 kinetochore localization in human cells.

The spindle assembly checkpoint is a surveillance mechanism that blocks anaphase onset until all chromosomes are properly attached to microtubules of the mitotic spindle. Checkpoint activity requires kinetochore localization of Mad1/Mad2 to inhibit activation of the anaphase promoting complex/cyclosome in the presence of unattached kinetochores. In budding yeast and Caenorhabditis elegans, Bub1, recruited to kinetochores through KNL1, recruits Mad1/Mad2 by direct linkage with Mad1. However, in human cells it is not yet established which kinetochore protein(s) function as the Mad1/Mad2 receptor. Both Bub1 and the RZZ complex have been implicated in Mad1/Mad2 kinetochore recruitment; however, their specific roles remain unclear. Here, we investigate the contributions of Bub1, RZZ and KNL1 to Mad1/Mad2 kinetochore recruitment. We find that the RZZ complex localizes to the N-terminus of KNL1, downstream of Bub1, to mediate robust Mad1/Mad2 kinetochore localization. Our data also point to the existence of a KNL1-, Bub1-independent mechanism for RZZ and Mad1/Mad2 kinetochore recruitment. Based on our results, we propose that in humans, the primary mediator for Mad1/Mad2 kinetochore localization is the RZZ complex.

Cell Division: Molecular Pathways for KMN Kinetochore Recruitment.

Robust but dynamic attachment between kinetochores and spindle microtubules is an essential prerequisite for accurate chromosome segregation and for preventing aneuploidy. A pair of recent studies has shed light on the details of how the molecular machinery that orchestrates these attachments is recruited to mitotic kinetochores.