Case report: isolation of Hydrogenophaga from septic blood culture following near-death drowning in lakewater.

A patient suffered a non-fatal wet drowning in a freshwater lake and developed bacteraemia several days later. Blood culture grew a Gram-negative rod that could not be identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). 16S ribosomal RNA sequencing of the isolate identified the microbe as Hydrogenophaga laconesensis - an environmental microbe commonly found in freshwater. The recovery of multiple pathogenic micro-organisms (although not H. laconesensis ) from culture of respiratory specimens prompted the initiation of antibiotic therapy with cefepime and, later, vancomycin. The patient's clinical course gradually improved over the course of 2 weeks and she was ultimately discharged home with minimal sequelae. To our knowledge, this is the first evidence of human infection with bacteria in the genus Hydrogenophaga . Hydrogenophaga may be considered in cases of freshwater near-drowning, and MALDI-TOF MS databases should be updated to include H. laconesensis .

Detection of Pneumocystis jirovecii from Clinical Specimens Utilizing a TaqMan-Based Real-Time PCR Assay on the Luminex ARIES.

Pneumocystis jirovecii can cause severe pneumonia in immunocompromised patients, which can be life threatening if left untreated. Despite the widespread use of polymerase chain reaction (PCR) within the clinical laboratory setting, FDA-approved PCR assays are not readily available for the detection of Pneumocystis from respiratory samples. Using the Luminex ARIES system-an open-channel, automated, sample-to-answer PCR platform-the cell division cycle 2 (cdc-2) gene can be targeted for the detection of Pneumocystis. This novel TaqMan-based, real-time PCR assay offers improved sensitivity compared to staining or immunofluorescence while reducing turnaround time and eliminating the challenges surrounding microscopic identification. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Primer/probe master mix preparation Basic Protocol 2: Positive control (cdc-2) plasmid preparation Basic Protocol 3: Mucus digestion Basic Protocol 4: Cell lysis Basic Protocol 5: Carrier RNA/proteinase K preparation Basic Protocol 6: Cassette assembly Basic Protocol 7: Running the assay Basic Protocol 8: Interpreting results.

Comparative study of Revogene GBS LB assay and GeneXpert GBS LB assay for the detection of group B Streptococcus in prenatal screening samples.

BACKGROUND: Group B Streptococcal (GBS) infections in the United States are a leading cause of meningitis and sepsis in newborns. The CDC therefore recommends GBS screening for all pregnant women at 35-37 weeks of gestation and administration of intrapartum prophylaxis (in those that tested positive) as an effective means of controlling disease transmission. Several FDA approved molecular diagnostic tests are available for rapid and accurate detection of GBS in antepartum women. METHOD: In this study, we report a clinical comparison of the Xpert GBS LB assay and a novel FDA-cleared test, Revogene GBS LB assay. A total of 250 vaginal-rectal swabs from women undergoing prenatal screening were submitted to the University of Wisconsin's clinical microbiology laboratory for GBS testing. RESULTS: We found 96.8% of samples were concordant between the two tests, while 3.2% were discordant with a positive percent agreement of 98.0% and a negative percent agreement of 96.5% between the Revogene GBS LB assay and the GeneXpert GBS LB assay. CONCLUSION: Overall, we report that both assays perform well for the detection of GBS colonization in pregnant women.

Streptococcus agalactiae Strains with Chromosomal Deletions Evade Detection with Molecular Methods.

Surveillance of circulating microbial populations is critical for monitoring the performance of a molecular diagnostic test. In this study, we characterized 31 isolates of Streptococcus agalactiae (group B Streptococcus [GBS]) from several geographic locations in the United States and Ireland that contain deletions in or adjacent to the region of the chromosome that encodes the hemolysin gene cfb, the region targeted by the Xpert GBS and GBS LB assays. PCR-negative, culture-positive isolates were recognized during verification studies of the Xpert GBS assay in 12 laboratories between 2012 and 2018. Whole-genome sequencing of 15 GBS isolates from 11 laboratories revealed four unique deletions of chromosomal DNA ranging from 181 bp to 49 kb. Prospective surveillance studies demonstrated that the prevalence of GBS isolates containing deletions in the convenience sample was <1% in three geographic locations but 7% in a fourth location. Among the 15 isolates with chromosomal deletions, multiple pulsed-field gel electrophoresis types were identified, one of which appears to be broadly dispersed across the United States.

Selenium and vitamin E for prostate cancer: post-SELECT (Selenium and Vitamin E Cancer Prevention Trial) status.

Various formulations of selenium and vitamin E, both essential human dietary components, have been shown to possess a therapeutic and preventive effect against prostate cancer. Fortuitous results of clinical trials also implied a risk-reduction effect of selenium and vitamin E supplements. The Selenium and Vitamin E Cancer Prevention Trial (SELECT), using oral selenium and vitamin E supplementation in disease-free volunteers, was designed to test a prostate cancer chemoprevention hypothesis. SELECT was terminated early because of both safety concerns and negative data for the formulations and doses given. Here, we review and discuss the studies done before and since the inception of SELECT, as well as the parameters of the trial itself. We believe that there is a lack of appropriate in vivo preclinical studies on selenium and vitamin E despite many promising in vitro studies on these agents. It seems that the most effective doses and formulations of these agents for prostate cancer chemoprevention have yet to be tested. Also, improved understanding of selenium and vitamin E biology may facilitate the discovery of these doses and formulations.

Melatonin, a novel Sirt1 inhibitor, imparts antiproliferative effects against prostate cancer in vitro in culture and in vivo in TRAMP model.

We recently demonstrated that Sirt1, a NAD(+) -dependent histone deacetylase, was overexpressed in prostate cancer (PCa) and its inhibition resulted in a significant antiproliferative response in human PCa cells. Studies have suggested a link between Sirt1 and circadian rhythms, the disruption of which has been linked to cancer. Interestingly, a decreased production of the pineal melatonin has been shown to deregulate the circadian rhythm machinery and increase cancer risk. Furthermore, disruption in melatonin production and circadian rhythmicity has been associated with aging. Here, we challenged our hypothesis that melatonin will impart antiproliferative response against PCa via inhibiting Sirt1. We demonstrated that melatonin significantly inhibited Sirt1 protein and activity in vitro in multiple human PCa cell lines, and melatonin-mediated Sirt1 inhibition was accompanied with a significant decrease in the proliferative potential of PCa cells, but not of normal cells. Forced overexpression of Sirt1 partially rescued the PCa cells from melatonin's antiproliferative effects, suggesting that Sirt1 is a direct target of melatonin. Employing transgenic adenocarcinoma of mouse prostate (TRAMP) mice, we also demonstrated that oral administration of melatonin, at human-achievable doses, significantly inhibited PCa tumorigenesis as shown by decreases in (i) prostate and genitourinary weight, (ii) serum insulin-like growth factor-1 (IGF-1)/IGF-binding protein-3 (IGFBP3) ratio, (iii) mRNA and protein levels of the proliferation markers (PCNA, Ki-67). This anti-PCa response was accompanied with a significant decrease in Sirt1 in TRAMP prostate. Our data identified melatonin as a novel inhibitor of Sirt1 and suggest that melatonin can inhibit PCa growth via Sirt1 inhibition.

Melatonin resynchronizes dysregulated circadian rhythm circuitry in human prostate cancer cells.

Prostate cancer (PCa) is a major age-related malignancy as increasing age correlates with increased risk for developing this neoplasm. Similarly, alterations in circadian rhythms have also been associated with the aging population and cancer risk. The pineal hormone melatonin is known to regulate circadian rhythms, which is under the control of a core set of genes: Period 1, 2, 3 (Per 1-3); Cryptochrome 1, 2 (Cry 1, 2); Clock, and Bmal 1, 2. Melatonin levels have been shown to decrease in patients with cancer and exogenous melatonin exhibits antiproliferative effects against certain cancers. In this study, we challenged the hypothesis that melatonin imparts antiproliferative effects in prostate cancer via resynchronization of deregulated core clock circuitry. We found that Clock and Per2 protein levels were downregulated whereas Bmal1 protein levels were upregulated in PCa cells, compared to normal prostate cells. Additionally, employing automated quantitative analysis of a microarray containing human tissues, we found that compared to benign tissues, Clock and Per2 levels were downregulated, whereas Bmal1 levels were upregulated in PCa and other proliferative prostatic conditions. Overexpression of Per2 was found to result in a significant loss of PCa cell growth and viability. Interestingly, melatonin treatment resulted in an increase in Per2 and Clock and a reduction in Bmal1 in PCa cells. Further, melatonin treatment resulted in a resynchronization of oscillatory circadian rhythm genes (Dbp and Per2). Our data support our hypothesis and suggest that melatonin should be thoroughly investigated as an agent for the management of PCa and other age-related malignancies.

Sirtuins, melatonin and circadian rhythms: building a bridge between aging and cancer.

Histone deacetylases (HDAC) have been under intense scientific investigation for a number of years. However, only recently the unique class III HDAC, sirtuins, have gained increasing investigational momentum. Originally linked to longevity in yeast, sirtuins and more specifically, SIRT1 have been implicated in numerous biological processes having both protective and/or detrimental effects. SIRT1 appears to play a critical role in the process of carcinogenesis, especially in age-related neoplasms. Similarly, alterations in circadian rhythms as well as production of the pineal hormone melatonin have been linked to aging and cancer risk. Melatonin has been found act as a differentiating agent in some cancer cells and to lower their invasive and metastatic status. In addition, melatonin synthesis and release occurs in a circadian rhythm fashion and it has been linked to the core circadian machinery genes (Clock, Bmal1, Periods, and Cryptochromes). Melatonin has also been associated with chronotherapy, the timely administration of chemotherapy agents to optimize trends in biological cycles. Interestingly, a recent set of studies have linked SIRT1 to the circadian rhythm machinery through direct deacetylation activity as well as through the nicotinamide adenine dinucleotide (NAD(+)) salvage pathway. In this review, we provide evidence for a possible connection between sirtuins, melatonin, and the circadian rhythm circuitry and their implications in aging, chronomodulation, and cancer.

SIRT1 controls circadian clock circuitry and promotes cell survival: a connection with age-related neoplasms.

Aging is believed to be a primary risk factor for cancer. Interestingly, the sirtuin family of class III histone deacetylases (HDACs) has been implicated in the regulation of longevity and may be a lost link between aging and cancer. SIRT1, a nicotinamide adenine dinucleotide (NAD(+))-dependent sirtuin, has been shown to promote cell survival by inhibiting apoptosis or cellular senescence in mammalian cells. Recent studies have provided a link between the cellular metabolic function of SIRT1 and the circadian rhythm (controlled by a clock machinery), which, if deregulated, may lead to an increased risk for some cancers. Interestingly, the loss of the pineal hormone melatonin, a known regulator of circadian rhythm, has been shown to cause deregulation in the circadian rhythm machinery and an increase in susceptibility to cancer. On the basis of scientific evidence, we propose a hypothesis that SIRT1 inhibition will impart an antiproliferative response in age-related cancers via resynchronization of deregulated core clock circuitry at the cellular level. If this hypothesis is found valid, it may ultimately lead to the development of novel approaches toward management of age-related malignancies and possibly other diseases.

Role of p53 in the anti-proliferative effects of Sirt1 inhibition in prostate cancer cells.

Prostate cancer (PCa), next only to skin cancer, is the most commonly occurring malignancy in men in the US. Aging is recognized as a major risk factor for this neoplasm as a man's chance for developing this disease significantly increases with increasing age. Because aging is inevitable, Americans are living longer, and the existing treatments have not been able to manage this neoplasm, novel mechanism-based approaches are needed. We have recently shown that Sirt1, a sirtuin class III histone deacetylases (HDACs) originally linked to aging and longevity in yeast, was overexpressed in human PCa cells and PCa tissues obtained from patients. We also found that chemical inhibition and/or genetic knockdown of Sirt1 caused a FoxO1-mediated inhibition in the growth and viability of human PCa cells. Since p53 is a target for deacetylation by Sirt1, we wanted to determine the involvement of p53 in Sirt1 inhibition mediated responses in PCa. To achieve our objective, we utilized a pair of isogenic PCa cell lines viz. PC3 and PC3-p53, which differ only in p53 status. Our data demonstrated that Sirt1 inhibition caused a decrease in cell growth, cell viability and the colony formation ability of both cell lines. Further, Sirt1 inhibition resulted in an increase in FoxO1 acetylation and subsequent transcriptional activation in both cell types regardless of p53 status. However, an interesting observation of our study was that Sirt1 inhibition resulted in an increase in senescence in PC3-p53 cells whereas it resulted in an increase in apoptosis in PC3 cells. The results of this study compliment our previous study and suggest that Sirt1 inhibition may have different downstream targets in cells with active p53 versus cells where p53 is inactive.

Role of sirtuin histone deacetylase SIRT1 in prostate cancer. A target for prostate cancer management via its inhibition?

Prostate cancer (PCa) is a major age-related malignancy, and according to estimates from the American Cancer Society, a man's chance of developing this cancer significantly increases with increasing age, from 1 in 10,149 by age 39 to 1 in 38 by age 59 to 1 in 7 by age 70. Therefore, it is important to identify the causal connection between mechanisms of aging and PCa. Employing in vitro and in vivo approaches, in this study, we tested the hypothesis that SIRT1, which belongs to the Sir2 (silent information regulator 2) family of sirtuin class III histone deacetylases, is overexpressed in PCa, and its inhibition will have antiproliferative effects in human PCa cells. Our data demonstrated that SIRT1 was significantly overexpressed in human PCa cells (DU145, LNCaP, 22Rnu1, and PC3) compared with normal prostate epithelial cells (PrEC) at protein, mRNA, and enzymatic activity levels. SIRT1 was also found to be overexpressed in human PCa tissues compared with adjacent normal prostate tissue. Interestingly, our data demonstrated that SIRT1 inhibition via nicotinamide and sirtinol (at the activity level) as well as via short hairpin RNA-mediated RNA interference (at the genetic level) resulted in a significant inhibition in the growth and viability of human PCa cells while having no effect on normal prostate epithelial cells. Further, we found that inhibition of SIRT1 caused an increase in FOXO1 acetylation and transcriptional activation in PCa cells. Our data suggested that SIRT1, via inhibiting FOXO1 activation, could contribute to the development of PCa. We suggest that SIRT1 could serve as a target toward developing novel strategies for PCa management.