Pancreatectomy Induces Cancer-Promoting Neutrophil Extracellular Traps.

BACKGROUND: Neutrophil extracellular traps (NETs) occur when neutrophil chromatin is decondensed and extruded into the extracellular space in a web-like structure. Originally described as an anti-microbial function, this process has been implicated in the pathogenesis of pancreatic disease. In addition, NETs are upregulated during physiologic wound-healing and coagulation. This study evaluated how the inflammatory response to pancreatic surgery influences NET formation. METHODS: For this study, 126 patients undergoing pancreatectomy gave consent before participation. Plasma was collected at several time points (preoperatively and through the postoperative outpatient visit). Plasma levels of NET markers, including cell-free DNA (cfDNA), citrullinated histone H3 (CitH3), interleukin (IL)-8, IL-6, and granulocyte colony-stimulating factor (G-CSF) were measured using enzyme-linked immunosorbent assay (ELISA). Patient clinical data were retrospectively collected from a prospectively maintained database. RESULTS: After pancreatic resection, NET markers (cfDNA and CitH3) were elevated, peaking on postoperative days 3 and 4. This increase in NETs was due to an inherent change in neutrophil biology. Postoperatively, NET-inducing cytokines (IL-8, IL-6, and G-CSF) were increased, peaking early in the postoperative course. The patients undergoing the robotic approach had a reduction in NETs during the postoperative period compared with those who underwent the open approach. The patients who experienced a pancreatic leak had an increase in NET markers during the postoperative period. CONCLUSIONS: Pancreatectomy induces cancer-promoting NET formation. The minimally invasive robotic approach may induce fewer NETs, although the current analysis was limited by selection bias. Pancreatic leak resulted in increased NETs. Further study into the potential for NET inhibition during the perioperative period is warranted.

SNAP-25 in Major Psychiatric Disorders: A Review.

Synaptosomal Associated Protein-25 kilodaltons (SNAP-25) is an integral member of the SNARE complex. This complex is essential for calcium-triggered synaptic vesicular fusion and release of neurotransmitters into the synaptic cleft. In addition to neurotransmission, SNAP-25 is associated with insulin release, the regulation of intracellular calcium, and neuroplasticity. Because of SNAP-25's varied and crucial biological roles, the consequences of changes in this protein can be seen in both the central nervous system and the periphery. In this review, we will look at the published literature from human genetic, postmortem, and animal studies involving SNAP-25. The accumulated data indicate that SNAP-25 may be linked with some symptoms associated with a variety of psychiatric disorders. These disorders include bipolar disorder, schizophrenia, major depressive disorder, attention deficit hyperactivity disorder, autism, alcohol use disorder, and dementia. There are also data suggesting SNAP-25 may be involved with non-psychiatric seizures and metabolic disorders. We believe investigation of SNAP-25 is important for understanding both normal behavior and some aspects of the pathophysiology of behavior seen with psychiatric disorders. The wealth of information from both animal and human studies on SNAP-25 offers an excellent opportunity to use a bi-directional research approach. Hypotheses generated from genetically manipulated mice can be directly tested in human postmortem tissue, and, conversely, human genetic and postmortem findings can improve and validate animal models for psychiatric disorders.

The human jejunum has an endogenous microbiota that differs from those in the oral cavity and colon.

BACKGROUND: The upper half of the human small intestine, known as the jejunum, is the primary site for absorption of nutrient-derived carbohydrates, amino acids, small peptides, and vitamins. In contrast to the colon, which contains 1011-1012 colony forming units of bacteria per ml (CFU/ml), the normal jejunum generally ranges from 103 to 105 CFU per ml. Because invasive procedures are required to access the jejunum, much less is known about its bacterial microbiota. Bacteria inhabiting the jejunal lumen have been investigated by classical culture techniques, but not by culture-independent metagenomics. RESULTS: The lumen of the upper jejunum was sampled during enteroscopy of 20 research subjects. Culture on aerobic and anaerobic media gave live bacterial counts ranging from 5.8 × 103 CFU/ml to 8.0 × 106 CFU/ml. DNA from the same samples was analyzed by 16S rRNA gene-specific quantitative PCR, yielding values from 1.5 × 105 to 3.1 × 107 bacterial genomes per ml. When calculated for each sample, estimated bacterial viability ranged from effectively 100% to a low of 0.3%. 16S rRNA metagenomic analysis of uncultured bacteria by Illumina MiSeq sequencing gave detailed microbial composition by phylum, genus and species. The genera Streptococcus, Prevotella, Veillonella and Fusobacterium, were especially abundant, as well as non-oral genera including Escherichia, Klebsiella, and Citrobacter. The jejunum was devoid of the genera Alistipes, Ruminococcus, Faecalibacterium, and other extreme anaerobes abundant in the colon. In patients with higher bacterial loads, there was no significant change in microbial species composition. CONCLUSIONS: The jejunal lumen contains a distinctive bacterial population consisting primarily of facultative anaerobes and oxygen-tolerant obligate anaerobes similar to those found in the oral cavity. However, the frequent abundance of Enterobacteriaceae represents a major difference from oral microbiota. Although a few genera are shared with the colon, we found no evidence for retrograde movement of the most abundant colonic microbes to the jejunum. Some individuals had much higher bacterial loads, but this was not correlated with decreases in bacterial species diversity or other evidence of dysbiosis.

Human embryonic stem cells have constitutively active Bax at the Golgi and are primed to undergo rapid apoptosis.

Human embryonic stem (hES) cells activate a rapid apoptotic response after DNA damage but the underlying mechanisms are unknown. A critical mediator of apoptosis is Bax, which is reported to become active and translocate to the mitochondria only after apoptotic stimuli. Here we show that undifferentiated hES cells constitutively maintain Bax in its active conformation. Surprisingly, active Bax was maintained at the Golgi rather than at the mitochondria, thus allowing hES cells to effectively minimize the risks associated with having preactivated Bax. After DNA damage, active Bax rapidly translocated to the mitochondria by a p53-dependent mechanism. Interestingly, upon differentiation, Bax was no longer active, and cells were not acutely sensitive to DNA damage. Thus, maintenance of Bax in its active form is a unique mechanism that can prime hES cells for rapid death, likely to prevent the propagation of mutations during the early critical stages of embryonic development.

Viral single-strand DNA induces p53-dependent apoptosis in human embryonic stem cells.

Human embryonic stem cells (hESCs) are primed for rapid apoptosis following mild forms of genotoxic stress. A natural form of such cellular stress occurs in response to recombinant adeno-associated virus (rAAV) single-strand DNA genomes, which exploit the host DNA damage response for replication and genome persistence. Herein, we discovered a unique DNA damage response induced by rAAV transduction specific to pluripotent hESCs. Within hours following rAAV transduction, host DNA damage signaling was elicited as measured by increased gamma-H2AX, ser15-p53 phosphorylation, and subsequent p53-dependent transcriptional activation. Nucleotide incorporation assays demonstrated that rAAV transduced cells accumulated in early S-phase followed by the induction of apoptosis. This lethal signaling sequalae required p53 in a manner independent of transcriptional induction of Puma, Bax and Bcl-2 and was not evident in cells differentiated towards a neural lineage. Consistent with a lethal DNA damage response induced upon rAAV transduction of hESCs, empty AAV protein capsids demonstrated no toxicity. In contrast, DNA microinjections demonstrated that the minimal AAV origin of replication and, in particular, a 40 nucleotide G-rich tetrad repeat sequence, was sufficient for hESC apoptosis. Our data support a model in which rAAV transduction of hESCs induces a p53-dependent lethal response that is elicited by a telomeric sequence within the AAV origin of replication.

SKPs derive from hair follicle precursors and exhibit properties of adult dermal stem cells.

Despite the remarkable regenerative capacity of mammalian skin, an adult dermal stem cell has not yet been identified. Here, we investigated whether skin-derived precursors (SKPs) might fulfill such a role. We show that SKPs derive from Sox2(+) hair follicle dermal cells and that these two cell populations are similar with regard to their transcriptome and functional properties. Both clonal SKPs and endogenous Sox2(+) cells induce hair morphogenesis, differentiate into dermal cell types, and home to a hair follicle niche upon transplantation. Moreover, hair follicle-derived SKPs self-renew, maintain their multipotency, and serially reconstitute hair follicles. Finally, grafting experiments show that follicle-associated dermal cells move out of their niche to contribute cells for dermal maintenance and wound-healing. Thus, SKPs derive from Sox2(+) follicle-associated dermal precursors and display functional properties predicted of a dermal stem cell, contributing to dermal maintenance, wound-healing, and hair follicle morphogenesis.

SOX2 is a dose-dependent regulator of retinal neural progenitor competence.

Approximately 10% of humans with anophthalmia (absent eye) or severe microphthalmia (small eye) show haploid insufficiency due to mutations in SOX2, a SOXB1-HMG box transcription factor. However, at present, the molecular or cellular mechanisms responsible for these conditions are poorly understood. Here, we directly assessed the requirement for SOX2 during eye development by generating a gene-dosage allelic series of Sox2 mutations in the mouse. The Sox2 mutant mice display a range of eye phenotypes consistent with human syndromes and the severity of these phenotypes directly relates to the levels of SOX2 expression found in progenitor cells of the neural retina. Retinal progenitor cells with conditionally ablated Sox2 lose competence to both proliferate and terminally differentiate. In contrast, in Sox2 hypomorphic/null mice, a reduction of SOX2 expression to <40% of normal causes variable microphthalmia as a result of aberrant neural progenitor differentiation. Furthermore, we provide genetic and molecular evidence that SOX2 activity, in a concentration-dependent manner, plays a key role in the regulation of the NOTCH1 signaling pathway in retinal progenitor cells. Collectively, these results show that precise regulation of SOX2 dosage is critical for temporal and spatial regulation of retinal progenitor cell differentiation and provide a cellular and molecular model for understanding how hypomorphic levels of SOX2 cause retinal defects in humans.

SOX2, a persistent marker for multipotential neural stem cells derived from embryonic stem cells, the embryo or the adult.

Multipotent neural stem cells are present throughout the development of the central nervous system (CNS), persist into adulthood in defined locations and can be derived from more primitive embryonic stem cells. We show that SOX2, an HMG box transcription factor, is expressed in multipotent neural stem cells at all stages of mouse ontogeny. We have generated transgenic mice expressing enhanced green fluorescent protein (EGFP) under the control of the endogenous locus-regulatory regions of the Sox2 gene to prospectively identify neural stem/progenitor cells in vivo and in vitro. Fluorescent cells coexpress SOX2 protein, and EGFP fluorescence is detected in proliferating neural progenitor cells of the entire anterior-posterior axis of the CNS from neural plate stages to adulthood. SOX2-EGFP cells can form neurospheres that can be passaged repeatedly and can differentiate into neurons, astrocytes and oligodendrocytes. Moreover, prospective clonal analysis of SOX2-EGFP-positive cells shows that all neurospheres, whether isolated from the embryonic CNS or the adult CNS, express SOX2-EGFP. In contrast, the pattern of SOX2-EGFP expression using randomly integrated Sox2 promoter/reporter construct differs, and neurospheres are heterogeneous for EGFP expression. These studies demonstrate that SOX2 may meet the requirements of a universal neural stem cell marker and provides a means to identify cells which fulfill the basic criteria of a stem cell: self-renewal and multipotent differentiation.