Role of Hypoxia in the Control of the Cell Cycle.

The cell cycle is an important cellular process whereby the cell attempts to replicate its genome in an error-free manner. As such, mechanisms must exist for the cell cycle to respond to stress signals such as those elicited by hypoxia or reduced oxygen availability. This review focuses on the role of transcriptional and post-transcriptional mechanisms initiated in hypoxia that interface with cell cycle control. In addition, we discuss how the cell cycle can alter the hypoxia response. Overall, the cellular response to hypoxia and the cell cycle are linked through a variety of mechanisms, allowing cells to respond to hypoxia in a manner that ensures survival and minimal errors throughout cell division.

Phenotypes, transcriptome, and novel biofilm formation associated with the ydcI gene.

The ydcI gene has previously been shown to encode a DNA-binding protein involved with acid stress resistance and induced biofilm formation in a strain of Salmonella enterica serovar Typhimurium. In addition, characterisation of the ydcI gene in Escherichia coli and other bacteria demonstrated strikingly different tolerance for induced ydcI expression across Gram negative species. In this report, we investigated the conservation of these phenotypes across multiple strains of S. Typhimurium and E. coli, and we used RNA Seq to identify the transcriptome of the ΔydcI mutant compared to WT in S. Typhimurium and E. coli (to establish the YdcI regulon in each species). We constructed deletion mutants in each species based on the RNA Seq results and tested these mutants for the relevant ydcI-related phenotypes. Though no evidence for a role in these phenotypes was found via the RNA Seq deletion mutants, we found that the ydcI-induced biofilm in S. Typhimurium is formed independently of the major biofilm genes csgA and bcsA indicating a potentially novel type of biofilm formation.

A Survey of Sleep Medicine Physician Perceptions on the Surgical Treatment of Obstructive Sleep Apnea.

PURPOSE: Surgical treatment for obstructive sleep apnea (OSA) varies by specialty. Our survey sought to answer 3 principal questions: 1) To which surgical specialists are sleep physicians referring patients for upper airway surgery? 2) Which surgical treatment do sleep specialists find to be most effective in treating OSA? 3) Do sleep medicine physicians believe that maxillomandibular advancement (MMA) is worthwhile to patients who are surgical candidates? MATERIALS AND METHODS: We formulated a cross-sectional survey. The study sample was obtained by identifying all practices that advertised as sleep medicine specialists in Houston, Texas, by using Internet searches. Physicians who treated children were excluded. Seventy-nine surveys were hand delivered to offices in the greater Houston area; the survey included 6 questions to determine referral and surgical preferences for OSA. Variable responses included years in practice, specialty, and a comments section. A 10-point Likert scale was used to assess sleep medicine physicians' referral patterns and perceptions regarding surgical treatment of OSA. Numerical data were analyzed by calculating mean values and by dividing responses into "disagree" (<5), "neutral" (5), and "agree" (>5). RESULTS: Twenty-six surveys were returned. More sleep medicine physicians referred patients to ear, nose, and throat surgeons (52%) than to oral and maxillofacial surgeons (20%). MMA was viewed as the most effective surgery (72%), followed by "none" (16%), "other" (8%), and uvulopalatopharyngoplasty (4%). More respondents viewed the benefits versus risks as favorable for MMA (44%) than for uvulopalatopharyngoplasty (29%). CONCLUSIONS: The results of this survey show that sleep medicine physicians in the greater Houston area view MMA as the most favorable and effective surgical option for treating OSA. Although MMA was most often referred for, more respondents refer patients to ear, nose, and throat surgeons than to oral and maxillofacial surgeons for surgical management of OSA. Years in practice displayed no correlation in referral patterns or preference for type of OSA surgery.

The Bacterial iprA Gene Is Conserved across Enterobacteriaceae, Is Involved in Oxidative Stress Resistance, and Influences Gene Expression in Salmonella enterica Serovar Typhimurium.

UNLABELLED: The iprA gene (formerly known as yaiV or STM0374) is located in a two-gene operon in the Salmonella enterica serovar Typhimurium genome and is associated with altered expression during spaceflight and rotating-wall-vessel culture conditions that increase virulence. However, iprA is uncharacterized in the literature. In this report, we present the first targeted characterization of this gene, which revealed that iprA is highly conserved across Enterobacteriaceae We found that S Typhimurium, Escherichia coli, and Enterobacter cloacae ΔiprA mutant strains display a multi-log-fold increase in oxidative stress resistance that is complemented using a plasmid-borne wild-type (WT) copy of the S Typhimurium iprA gene. This observation was also associated with increased catalase activity, increased S Typhimurium survival in macrophages, and partial dependence on the katE gene and full dependence on the rpoS gene. Our results indicate that IprA protein activity is sensitive to deletion of the N- and C-terminal 10 amino acids, while a region that includes amino acids 56 to 80 is dispensable for activity. RNA sequencing (RNA-Seq) analysis revealed several genes altered in expression in the S Typhimurium ΔiprA mutant strain compared to the WT, including those involved in fimbria formation, spvABCD-mediated virulence, ethanolamine utilization, the phosphotransferase system (PTS) transport, and flagellin phase switching from FlgB to FliC (likely a stochastic event) and several genes of hypothetical or putative function. IMPORTANCE: Overall, this work reveals that the conserved iprA gene measurably influences bacterial biology and highlights the pool of currently uncharacterized genes that are conserved across bacterial genomes. These genes represent potentially useful targets for bacterial engineering, vaccine design, and other possible applications.

Bacterial genus-specific tolerance for YdcI expression.

The bacterial ydcI gene encodes a highly conserved transcriptional regulatory protein found in a wide range of Gram-negative bacteria and is involved in a number of Salmonella enterica serovar Typhimurium phenotypes. Given its high conservation, the YdcI protein has the potential for studies and applications across bacterial genera. However, no studies have been performed with YdcI outside of S. Typhimurium. Here we report that different Gram-negative genera display dramatically different tolerances for YdcI expression. In non-tolerant genera, YdcI expression results in rapid loss of cell viability several log-fold in magnitude, and the viability loss is observed at YdcI levels that are physiologically relevant. The N-terminal and C-terminal halves can be exchanged between the S. Typhimurium and Escherichia coli YdcI proteins with the resulting proteins still displaying the differential tolerance phenotype. Comparison of YdcI expression from the respective chromosomal gene in S. Typhimurium and E. coli revealed much lower levels in E. coli suggesting that this species has evolved a lower endogenous YdcI expression level and does not tolerate increases above this level. Expression of YdcI resulted in increased sensitivity to a range of antibiotics indicating the possibility that this protein could augment antibacterial strategies in non-tolerant genera. Overall, the results indicate vastly different outcomes for YdcI expression depending on bacterial genus and unmask differences in YdcI expression, regulation, target interactions, and/or YdcI regulon activity in different bacteria. The results also impact future work on YdcI when the protein is being studied/expressed in different Gram-negative genera.

Engineering large functional plasmids for biosafety.

Large bacterial plasmid constructs (generally 25-100 kb, but can be greater), such as those engineered with DNA encoding specific functions such as protein secretion or specialized metabolism, can carry antibiotic resistance genes and/or conjugation systems that typically must be removed before use in medical or environmental settings due to biosafety concerns. However, a convenient in vivo recombineering approach for intact large plasmids to sequentially remove multiple different genes using non-antibiotic selection methods is not described in the literature to our knowledge. We developed strategies and reagents for convenient removal of antibiotic resistance markers and conjugation genes while retaining non-antibiotic-based plasmid selection to increase practical utility of large engineered plasmids. This approach utilizes targeted lambda Red recombination of PCR products encoding the trpE and asd genes and as well as FLP/FRT-mediated marker removal. This is particularly important given that use of restriction enzymes with plasmids of this size is extremely problematic and often not feasible. This report provides the first example of the trpE gene/tryptophan prototrophy being used for recombineering selection. We applied this strategy to the plasmids R995+SPI-1 and R995+SPI-2 which encode cloned type III secretion systems to allow protein secretion and substrate delivery to eukaryotic cells. The resulting constructs are functional, stably maintained under conditions where the original constructs are unstable, completely defective for conjugative transfer, and transferred via electroporation.

Salmonella enterica serovar Typhimurium STM1266 encodes a regulator of curli biofilm formation: the brfS gene.

The major biofilm pathway in Salmonella enterica serovar Typhimurium involves specific growth conditions that induce the csgA gene whose product forms surface curli fibers that mediate biofilm formation. We have found that the previously uncharacterized STM1266 gene in S. Typhimurium plays a role in regulating biofilm formation via the curli pathway. S. Typhimurium ΔSTM1266 strains display a biofilm defect, and overexpression of STM1266 results in enhanced biofilm formation. STM1266 deletion resulted in lowered csgA expression using promoter-reporter ß-galactosidase assays, and csgA and csgD deletions abrogate the effects of STM1266 overexpression on biofilm formation while deletion of bcsA (encoding an essential enzyme for cellulose formation) has no effect. In a mouse infection model, the ΔSTM1266 strain displayed results similar to those seen for previously reported ΔcsgA strains. The STM1266 gene is predicted to encode a DNA-binding transcriptional regulator of the MerR family and is homologous to the Escherichia coli BluR regulator protein. We respectfully propose to ascribe the name brfS (biofilm regulator for Salmonella Typhimurium) to the STM1266 gene.

Characterization of the Salmonella enterica serovar Typhimurium ydcI gene, which encodes a conserved DNA binding protein required for full acid stress resistance.

Salmonella enterica serovar Typhimurium possesses a stimulon of genes that are differentially regulated in response to conditions of low fluid shear force that increase bacterial virulence and alter other phenotypes. In this study, we show that a previously uncharacterized member of this stimulon, ydcI or STM1625, encodes a highly conserved DNA binding protein with related homologs present in a range of gram-negative bacterial genera. Gene expression analysis shows that ydcI is expressed in different bacterial genera and is involved in its autoregulation in S. Typhimurium. We demonstrate that purified YdcI protein specifically binds a DNA probe consisting of its own promoter sequence. We constructed an S. Typhimurium ΔydcI mutant strain and show that this strain is more sensitive to both organic and inorganic acid stress than is an isogenic WT strain, and this defect is complemented in trans. Moreover, our data indicate that ydcI is part of the rpoS regulon related to stress resistance. The S. Typhimurium ΔydcI mutant was able to invade cultured cells to the same degree as the WT strain, but a strain in which ydcI expression is induced invaded cells at a level 2.8 times higher than that of the WT. In addition, induction of ydcI expression in S. Typhimurium resulted in the formation of a biofilm in stationary-phase cultures. These data indicate the ydcI gene encodes a conserved DNA binding protein involved with aspects of prokaryotic biology related to stress resistance and possibly virulence.

Transcriptional and proteomic responses of Pseudomonas aeruginosa PAO1 to spaceflight conditions involve Hfq regulation and reveal a role for oxygen.

Assessing bacterial behavior in microgravity is important for risk assessment and prevention of infectious diseases during spaceflight missions. Furthermore, this research field allows the unveiling of novel connections between low-fluid-shear regions encountered by pathogens during their natural infection process and bacterial virulence. This study is the first to characterize the spaceflight-induced global transcriptional and proteomic responses of Pseudomonas aeruginosa, an opportunistic pathogen that is present in the space habitat. P. aeruginosa responded to spaceflight conditions through differential regulation of 167 genes and 28 proteins, with Hfq as a global transcriptional regulator. Since Hfq was also differentially regulated in spaceflight-grown Salmonella enterica serovar Typhimurium, Hfq represents the first spaceflight-induced regulator acting across bacterial species. The major P. aeruginosa virulence-related genes induced in spaceflight were the lecA and lecB lectin genes and the gene for rhamnosyltransferase (rhlA), which is involved in rhamnolipid production. The transcriptional response of spaceflight-grown P. aeruginosa was compared with our previous data for this organism grown in microgravity analogue conditions using the rotating wall vessel (RWV) bioreactor. Interesting similarities were observed, including, among others, similarities with regard to Hfq regulation and oxygen metabolism. While RWV-grown P. aeruginosa mainly induced genes involved in microaerophilic metabolism, P. aeruginosa cultured in spaceflight presumably adopted an anaerobic mode of growth, in which denitrification was most prominent. Whether the observed changes in pathogenesis-related gene expression in response to spaceflight culture could lead to an alteration of virulence in P. aeruginosa remains to be determined and will be important for infectious disease risk assessment and prevention, both during spaceflight missions and for the general public.

Manipulating microcompartment operons to study mechanism and function.

The gene systems that encode functional bacterial microcompartments (BMCs) are typically comprised of between 10-23 genes, often in a contiguous operon. BMC genes can be studied as whole native operons or as subsets of genes that form structures for specific applications. Recent examples of such studies highlight the flexible modular nature of BMC operons/genes and the options that exist to harness their functions via manipulation at the DNA level. This work also demonstrates the transfer and functional expression of BMC operons/genes across bacterial species. Recombineering, DNA synthesis technology, and advanced cloning techniques have all been applied in creative ways to study the nature of BMC mechanism and function.

A series of vectors with alternative antibiotic resistance markers for use in lambda Red recombination.

A target bacterial strain of interest for use in Red-based recombineering may already encode resistance to antibiotic markers used with current Red recombination tools such that the resistance cannot be removed. Such cases include those where markers are needed to maintain an unstable genetic element co-resident in the strain or those where the genetic source of resistance is not known. We report the availability of PCR templates with FRT-flanked mutagenesis cassettes and plasmids encoding Red recombination functions that contain marker combinations not currently available on widely disseminated lambda Red molecular reagents. The functionality of these convenient alternative tools is demonstrated.

A series of IncQ-based reporter plasmids for use in a range of Gram negative genera.

Many studies require expression analysis of the same gene/promoter across a range of bacterial genera. However, there is currently a lack of availability of reporters based on the broad host range IncQ replicon, which is compatible with a popular improved IncP transfer system that is self-transfer defective. We report IncQ lacZ reporter plasmids with features including: (1) compatibility with IncP, IncW, and pBHR/pBBR replicons, (2) a variety of antibiotic markers (Sp-r, Sm-r, Km-r, Cm-r), (3) convenient mobilization via a novel self-transfer-defective IncP conjugation system, and (4) complete DNA sequences. Utility is demonstrated using three different promoters in different Gram negative genera.

Author Correction: Hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors induce autophagy and have a protective effect in an in-vitro ischaemia model.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors induce autophagy and have a protective effect in an in-vitro ischaemia model.

This study compared effects of five hypoxia-inducible factor (HIF) prolyl hydroxylases (PHD) inhibitors on PC12 cells and primary rat neurons following oxygen-glucose deprivation (OGD). At 100 µM, the PHD inhibitors did not cause cytotoxicity and apoptosis. MTT activity was only significantly reduced by FG4592 or Bayer 85-3934 in PC12 cells. The PHD inhibitors at 100 µM significantly increased the LC3-II/LC3-I expression ratio and downregulated p62 in PC12 cells, so did FG4592 (30 µM) and DMOG (100 µM) in neurons. HIF-1α was stabilised in PC12 cells by all the PHD inhibitors at 100 µM except for DMOG, which stabilised HIF-1α at 1 and 2 mM. In primary neurons, HIF-1α was stabilised by FG4592 (30 µM) and DMOG (100 µM). Pretreatment with the PHD inhibitors 24 hours followed by 24 hour reoxygenation prior to 6 hours OGD (0.3% O2) significantly reduced LDH release and increased MTT activity compared to vehicle (1% DMSO) pretreatment. In conclusion, the PHD inhibitors stabilise HIF-1α in normoxia, induce autophagy, and protect cells from a subsequent OGD insult. The new class of PHD inhibitors (FG4592, FG2216, GSK1278863, Bay85-3934) have the higher potency than DMOG. The interplay between autophagy, HIF stabilisation and neuroprotection in ischaemic stroke merits further investigation.

The cloned SPI-1 type 3 secretion system can be functionally expressed outside Salmonella backgrounds.

Due to its potential for use in bacterial engineering applications, we previously cloned the SPI-1 type 3 secretion system (T3SS) genes from the genome of Salmonella enterica serovar Typhimurium strain LT2. We have documented that this clone, while functionally expressed in S. Typhimurium strains, displays a severe expression defect in other Gram negative backgrounds including Escherichia coli. To address this issue, we compared SPI-1 DNA sequence across different backgrounds, fully sequenced the original SPI-1 clone, and cloned SPI-1 from other S. Typhimurium strains. In this process, we were able to successfully obtain SPI-1 clones that are functionally expressed in E. coli indicating the first such result for a full-length SP-1 T3SS clone. We discovered that the original cloning technique using a DNA homology-based capture method was the root of the expression defect and that the FRT-Capture technique is preferable over the homology-based method. This result paves the way for future studies and applications using cloned SPI-1 and other T3SS in non-Salmonella bacterial backgrounds.

Oxygen-sensing mechanisms in cells.

The importance of oxygen for the survival of multicellular and aerobic organisms is well established and documented. Over the years, increased knowledge of its use for bioenergetics has placed oxygen at the centre of research on mitochondria and ATP-generating processes. Understanding the molecular mechanisms governing cellular oxygen sensing and response has allowed for the discovery of novel pathways oxygen is involved in, culminating with the award of the Nobel Prize for Medicine and Physiology in 2019 to the pioneers of this field, Greg Semenza, Peter Ratcliffe and William Kaelin. However, it is now beginning to be appreciated that oxygen can be a signalling molecule involved in a vast array of molecular processes, most of which impinge on gene expression control. This review will focus on the knowns and unknowns of oxygen as a signalling molecule, highlighting the role of 2-oxoglutarate-dependent dioxygenases as central players in the cellular response to deviations in oxygen tension.

Celebrating 75 years.

INTRODUCTION: Following the introduction of shock wave lithotripsy (SWL), ureteroscopy (URS), and percutaneous nephrolithotomy (PCNL), the subspecialty of endourology was born in the late 1970s. The purpose of this study was to report milestones in Canadian endourology, highlighting Canada's contributions to the field. METHODS: A review of the literature was performed from the late 1970s to the present. The literature review included bibliographic and digital resources. Additionally, records and recollections by various individuals were used, including some who were directly involved. RESULTS: Endourology was born in Canada when SWL, URS, and PCNL emerged as minimally invasive treatment options for stones in the early to mid-1980s. According to our research, the first PCNL was performed at the University of Toronto in 1981. Dr. Joachim Burhenne, a Harvard-trained radiologist from Germany, first used extracorporeal SWL in Canada at the University of British Columbia (UBC) for the treatment of biliary stones. Treatment for urinary tract stones followed at UBC and Dalhousie University. The first worldwide use of the holmium laser for lithotripsy of urinary tract calculi took place at the University of Western Ontario. Other endourology milestones in Canada include the formation of the Canadian Endourology Group and the emergence of the Endourological Society-accredited fellowship programs at the University of Toronto and Western University in the 1990s. Canada hosted the 21st and 35th World Congress of Endourology and Shock Wave Lithotripsy annual meeting in Montreal and Vancouver, respectively. CONCLUSIONS: Canadian urologists have led many advances in SWL, URS, and PCNL over the past four decades and, for a relatively small community, have made significant contributions to the field. Through the training of the next generation of endourologists at Canadian institutions, the future of endourology in Canada is bright.

Differentially Marked IncP-1ß R751 Plasmids for Cloning via Recombineering and Conjugation.

We demonstrate here for the first time the use of an IncP-1ß plasmid, R751, as a gene capture vehicle for recombineering/conjugation strategies to clone large segments of bacterial genomes (20 - 100 + Kb). We designed R751 derivatives containing alternative markers for greater flexibility when using the R751 vehicle across different bacteria. These markers are removable if desired as part of the cloning procedure (with no extra steps needed). We demonstrated utility via cloning of 38 and 22 kb genomic segments from Salmonella enterica serovar Typhimurium and Escherichia coli, respectively. The plasmids expand the options available for use in recombineering/conjugation-based cloning applications.We demonstrate here for the first time the use of an IncP-1ß plasmid, R751, as a gene capture vehicle for recombineering/conjugation strategies to clone large segments of bacterial genomes (20 ­ 100 + Kb). We designed R751 derivatives containing alternative markers for greater flexibility when using the R751 vehicle across different bacteria. These markers are removable if desired as part of the cloning procedure (with no extra steps needed). We demonstrated utility via cloning of 38 and 22 kb genomic segments from Salmonella enterica serovar Typhimurium and Escherichia coli, respectively. The plasmids expand the options available for use in recombineering/conjugation-based cloning applications.

Hypoxia induces rapid changes to histone methylation and reprograms chromatin.

Oxygen is essential for the life of most multicellular organisms. Cells possess enzymes called molecular dioxygenases that depend on oxygen for activity. A subclass of molecular dioxygenases is the histone demethylase enzymes, which are characterized by the presence of a Jumanji-C (JmjC) domain. Hypoxia can alter chromatin, but whether this is a direct effect on JmjC-histone demethylases or due to other mechanisms is unknown. Here, we report that hypoxia induces a rapid and hypoxia-inducible factor-independent induction of histone methylation in a range of human cultured cells. Genomic locations of histone-3 lysine-4 trimethylation (H3K4me3) and H3K36me3 after a brief exposure of cultured cells to hypoxia predict the cell's transcriptional response several hours later. We show that inactivation of one of the JmjC-containing enzymes, lysine demethylase 5A (KDM5A), mimics hypoxia-induced cellular responses. These results demonstrate that oxygen sensing by chromatin occurs via JmjC-histone demethylase inhibition.