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In this study, we sought to profile the abundances and drivers of antibiotic resistance genes in an urban river impacted by combined sewage overflow (CSO) events. Water samples were collected weekly during the summer for two years; then, quantitative PCR was applied to determine the abundance of resistance genes associated with tetracycline, quinolones, and β-lactam antibiotics. In addition to sampling a CSO-impacted site near the city center, we also sampled a less urban site ~12 km upstream with no proximal sewage inputs. The tetracycline genes tetO and tetW were rarely found upstream, but were common at the CSO-impacted site, suggesting that the primary source was untreated sewage. In contrast, ampC was detected in all samples indicating a more consistent and diffuse source. The two other genes, qnrA and bla TEM , were present in only 40-50% of samples and showed more nuanced spatiotemporal patterns consistent with upstream agricultural inputs. The results of this study highlight the complex sources of ARGs in urban riverine ecosystems, and that interdisciplinary collaborations across diverse groups of stakeholders are necessary to combat the emerging threat of antibiotic resistance through anthropogenic pollution.

The spread of antibiotic-resistant bacteria (ARB) associated with wastewater is a significant environmental concern, but little is known about the persistence and proliferation of these organisms in receiving water bodies after discharge. To address this knowledge gap, we performed a series of microcosm experiments in which river water was amended with either untreated or treated wastewater, and the abundance of viable ciprofloxacin-, Bactrim-, and erythromycin-resistant bacteria was monitored for 72 h. Both types of wastewater amendments significantly increased the initial abundance of ARB compared to microcosms containing only river water (all p < 0.03). The increase was greatest with untreated wastewater, but that effect decreased steadily over time. In contrast, microcosms amended with treated wastewater saw a smaller initial increase and more complex temporal dynamics. Following a brief lag, ARB abundance bloomed for all three of the antibiotics that we considered. This suggests that ARB that survive wastewater treatment are particularly hardy and may proliferate in riverine conditions after a short recovery period. To determine how interactions with the native river microbial community impacted the persistence of wastewater-associated ARB, an additional set of microcosms was prepared using filter-sterilized river water. Peak abundance in these microcosms was significantly higher by 1-2 orders of magnitude compared to microcosms containing an intact river microbial community (all p < 0.05), which suggests that biotic interactions play a significant role in regulating the persistence and proliferation of ARB. The data presented in this paper are among the first available that specifically consider persistence of viable ARB and represent an important step toward understanding AR-related human health risks downstream from wastewater discharge points and following sewer overflow events. Additional studies that consider longer time scales and the interplay of biotic and abiotic variables are essential for modeling public health risks associated with wastewater inputs of ARB to rivers and other aquatic environments., Competing Interests: Declaration of competing interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Biology is an increasingly quantitative science. Thus, it is important that undergraduate biology curricula include frequent opportunities for students to practice their quantitative skills. This can create a substantial grading burden for faculty teaching online and/or large enrollment courses, but the "formula question" feature present in many learning management systems (LMS) offers a solution. Using this feature, faculty set up a basic scaffold for an algebraic word problem, and the LMS can then automatically generate and grade many different versions of the question. In this paper, we describe the use of "formula questions" in an undergraduate microbiology course and specifically focus on how the strategic use of algebraic word problems at multiple points throughout the semester can help build quantitative literacy. Key to the success of this approach is that faculty provide a review of foundational mathematical skills early in the semester, even in upper-level classes. This should include reacquainting students with formatting conventions (e.g., rounding and scientific notation), familiarizing them with any idiosyncrasies of the technology platforms, and demonstrating how to solve math problems using spreadsheets. This initial effort increases student success when more complex problems are introduced later in the semester. Though the tips summarized in this paper focus on undergraduate microbiology teaching laboratories using Canvas, the approach can easily be modified to help students develop their critical thinking and quantitative reasoning skills at other levels and in other disciplines., Competing Interests: The authors declare no conflict of interest., (Copyright © 2022 Battistelli and Franklin.)

In freshwater wetlands, competitive and cooperative interactions between respiratory, fermentative and methanogenic microbes mediate the decomposition of organic matter. These interactions may be disrupted by saltwater intrusion disturbances that enhance the activity of sulfate-reducing bacteria (SRB), intensifying their competition with syntrophic bacteria and methanogens for electron donors. We simulated saltwater intrusion into wetland soil microcosms and examined biogeochemical and microbial responses, employing metabolic inhibitors to isolate the activity of various microbial functional groups. Sulfate additions increased total carbon dioxide production but decreased methane production. Butyrate degradation assays showed continued (but lower) levels of syntrophic metabolism despite strong demand by SRB for this key intermediate decomposition product and a shift in the methanogen community toward acetoclastic members. One month after removing SRB competition, total methane production recovered by only ∼50%. Similarly, butyrate assays showed an altered accumulation of products (including less methane), although overall rates of syntrophic butyrate breakdown largely recovered. These effects illustrate that changes in carbon mineralization following saltwater intrusion are driven by more than the oft-cited competition between SRB and methanogens for shared electron donors. Thus, the impacts of disturbances on wetland biogeochemistry are likely to persist until cooperative and competitive microbial metabolic interactions can recover fully., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Soils are among the most biodiverse habitats on earth and while the species composition of microbial communities can influence decomposition rates and pathways, the functional significance of many microbial species and phylogenetic groups remains unknown. If bacteria exhibit phylogenetic organization in their function, this could enable ecologically meaningful classification of bacterial clades. Here, we show non-random phylogenetic organization in the rates of relative carbon assimilation for both rapidly mineralized substrates (amino acids and glucose) assimilated by many microbial taxa and slowly mineralized substrates (lipids and cellulose) assimilated by relatively few microbial taxa. When mapped onto bacterial phylogeny using ancestral character estimation this phylogenetic organization enabled the identification of clades involved in the decomposition of specific soil organic matter substrates. Phylogenetic organization in substrate assimilation could provide a basis for predicting the functional attributes of uncharacterized microbial taxa and understanding the significance of microbial community composition for soil organic matter decomposition., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

We investigated the effects of trace metal additions on microbial nitrogen (N) and carbon (C) cycling using freshwater wetland sediment microcosms amended with micromolar concentrations of copper (Cu), molybdenum (Mo), iron (Fe), and all combinations thereof. In addition to monitoring inorganic N transformations (NO 3 - , NO 2 - , N 2 O, NH 4 + ) and carbon mineralization (CO 2 , CH 4 ), we tracked changes in functional gene abundance associated with denitrification ( nirS , nirK , nosZ ), dissimilatory nitrate reduction to ammonium (DNRA; nrfA ), and methanogenesis ( mcrA ). With regards to N cycling, greater availability of Cu led to more complete denitrification (i.e., less N 2 O accumulation) and a higher abundance of the nirK and nosZ genes, which encode for Cu-dependent reductases. In contrast, we found sparse biochemical evidence of DNRA activity and no consistent effect of the trace metal additions on nrfA gene abundance. With regards to C mineralization, CO 2 production was unaffected, but the amendments stimulated net CH 4 production and Mo additions led to increased mcrA gene abundance. These findings demonstrate that trace metal effects on sediment microbial physiology can impact community-level function. We observed direct and indirect effects on both N and C biogeochemistry that resulted in increased production of greenhouse gasses, which may have been mediated through the documented changes in microbial community composition and shifts in functional group abundance. Overall, this work supports a more nuanced consideration of metal effects on environmental microbial communities that recognizes the key role that metal limitation plays in microbial physiology., (Copyright © 2020 Giannopoulos, Hartop, Brown, Song, Elsgaard and Franklin.)

Evidence is evolving that support the relationship that all carcinomas exhibit the following important relationships: The malignant cells exhibit a significant decreased zinc compared to the normal cells. The higher zinc levels that exist in the normal cells are cytotoxic in the malignant cells. The decrease in zinc is due to the down regulation of the ZIP-family zinc uptake transporter. These cells are as "ZIP-deficient/decreased zinc" malignancies. This provides a target for a chemotherapy that can restore the high zinc levels that will manifest cytotoxic effects in the malignant cells. In order to achieve this, a vehicle that facilitates the uptake and accumulation of zinc in the ZIP-deficient cells is required. The zinc ionophore, clioquinol, exhibits the properties that will provide these requirements. This is demonstrated by the treatment of a patient with 3% Clioquinol Cream, which successfully suppressed the progression of androgen-dependent prostate cancer. This treatment should also be efficacious for pancreatic cancer, liver cancer, breast cancer, thyroid cancer, kidney cancer, stomach cancer, gall bladder cancer, and lung cancer; which are carcinomas that exhibit decreased zinc. Thus, it is appropriate to describe that "Zinc is the wonder drug for the treatment of carcinomas".

Few studies have investigated bacterial community succession and the role of bacterial decomposition over a continuum of wood decay. Here, we identified how (i) the diversity and abundance of bacteria changed along a chronosequence of decay in Populus grandidentata (bigtooth aspen); (ii) bacterial community succession was dependent on the physical and chemical characteristics of the wood; (iii) interspecific bacterial interactions may mediate community structure. Four hundred and fifty-nine taxa were identified through Illumina sequencing of 16S rRNA amplicons from samples taken along a continuum of decay, representing standing dead trees, downed wood, and soil. Community diversity increased as decomposition progressed, peaking in the most decomposed trees. While a small proportion of taxa displayed a significant pattern in regards to decay status of the host log, many bacterial taxa followed a stochastic distribution. Changes in the water availability and chemical composition of standing dead and downed trees and soil were strongly coupled with shifts in bacterial communities. Nitrogen was a major driver of succession and nitrogen-fixing taxa of the order Rhizobiales were abundant early in decomposition. Recently downed logs shared 65% of their bacterial abundance with the microbiomes of standing dead trees while only sharing 16% with soil. As decay proceeds, bacterial communities appear to respond less to shifting resource availability and more to interspecific bacterial interactions - we report an increase in both the proportion (+9.3%) and the intensity (+62.3%) of interspecific interactions in later stages of decomposition, suggesting the emergence of a more complex community structure as wood decay progresses.

Studies have shown that a subgroup of tumor cells possess stemness characteristics having self-renewal capacity and the ability to form new tumors. We sought to identify the plausible stemness factor that determines the "molecular signature" of prostate cancer (PCa) cells derived from different metastases (PC3, PCa2b, LNCaP, and DU145) and whether androgen receptor (AR) influences the maintenance of stemness features. Here we show sex-determining region Y (SRY)-box 2 (SOX2) as a putative stem cell marker in PC3 PCa cells and not in DU145, PCa2b, or LNCaP cells. PCa2b and PC3 cells were derived from bone metastases. PCa2b cells which are positive for the AR failed to demonstrate the expression of either cluster of differentiation 44 (CD44) or SOX2. Knockdown (KD) of AR in these cells did not affect the expression of either CD44 or SOX2. Conversely, PC3 cells, which are negative for AR, expressed both CD44 and SOX2. However, the expression of AR downregulated the expression of both CD44 and SOX2 in PC3 cells. CD44 regulates SOX2 expression as KD of CD44 and reduces SOX2 levels considerably. SOX2 KD attenuated not only the expression of SNAIL and SLUG but also the migration and tumorsphere formation in PC3 cells. Collectively, our findings underscore a novel role of CD44 signaling in the maintenance of stemness and progression of cancer through SOX2 in AR-independent PC3 cells. SOX2 has a role in the regulation of expression of SNAIL and SLUG. SOX2 could be a potential therapeutic target to thwart the progression of SOX2-positive cancer cells or recurrence of androgen-independent PCa., (© 2018 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.)

Sea level rise and changes in precipitation can cause saltwater intrusion into historically freshwater wetlands, leading to shifts in microbial metabolism that alter greenhouse gas emissions and soil carbon sequestration. Saltwater intrusion modifies soil physicochemistry and can immediately affect microbial metabolism, but further alterations to biogeochemical processing can occur over time as microbial communities adapt to the changed environmental conditions. To assess temporal changes in microbial community composition and biogeochemical activity due to saltwater intrusion, soil cores were transplanted from a tidal freshwater marsh to a downstream mesohaline marsh and periodically sampled over 1 year. This experimental saltwater intrusion produced immediate changes in carbon mineralization rates, whereas shifts in the community composition developed more gradually. Salinity affected the composition of the prokaryotic community but did not exert a strong influence on the community composition of fungi. After only 1 week of saltwater exposure, carbon dioxide production doubled and methane production decreased by three orders of magnitude. By 1 month, carbon dioxide production in the transplant was comparable to the saltwater controls. Over time, we observed a partial recovery in methane production which strongly correlated with an increase in the relative abundance of three orders of hydrogenotrophic methanogens. Taken together, our results suggest that ecosystem responses to saltwater intrusion are dynamic over time as complex interactions develop between microbial communities and the soil organic carbon pool. The gradual changes in microbial community structure we observed suggest that previously freshwater wetlands may not experience an equilibration of ecosystem function until long after initial saltwater intrusion. Our results suggest that during this transitional period, likely lasting years to decades, these ecosystems may exhibit enhanced greenhouse gas production through greater soil respiration and continued methanogenesis., (© 2018 John Wiley & Sons Ltd.)

Introduction: Testosterone promotes the initial development of androgen-dependent prostate cancer. This is the basis for androgen ablation treatment, which attenuates, but does not terminate, the malignancy. Instead, it leads to prolactin-dependent malignancy; in which patient death generally occurs within 5 years. This report describes the novel treatment of a patient; which terminated androgen-independent prostate cancer., Results: Patient "XY" was diagnosed with prostate malignancy and metastases. He received hormonal androgen ablation treatment, chemotherapy, and radiation treatment. He developed androgen-independent prostate cancer; with expected death in 2-3 years. He was treated with cabergoline (dopamine agonist) treatment, which decreased the plasma prolactin 88%; by inhibiting the pituitary production of prolactin. The subsequent PET scan (positron emission tomography) revealed the absence of malignancy; and the CTC (circulating tumor cells) decreased from count=5.4 to count=0., Discussion: The cause of androgen-independent malignancy has been unknown, and an effective chemotherapy did not exist. The activities of normal and malignant prostate cells are regulated primarily by testosterone. When testosterone availability diminishes; prolactin regulation is manifested. This is represented when androgen ablation results in the development of prolactin-dependent malignancy. An effective chemotherapy would be targeted to eliminate the plasma prolactin-manifestation of the androgen-independent malignancy., Conclusions: This report of a novel chemotherapy for androgen-independent malignancy corroborates our understanding of the implications of prolactin in its development and treatment. There are about 165,000 cases/year with 25,000 deaths/year in the U.S.; and 1.0 million cases/year with 260,000 deaths/year worldwide. Those patients with androgen-independent prostate cancer can now employ this cabergoline treatment to prevent or terminate this deadly type of prostate cancer.

All cases of prostate cancer exhibit the hallmark condition of marked decrease in zinc in malignancy compared to the high zinc levels in the normal and benign prostate. There exists no reported corroborated case of prostate cancer in which malignancy exhibits the high zinc levels that exist in the normal prostate acinar epithelium. The decrease in zinc is achieved by the downregulation of ZIP1 zinc transporter, which prevents the uptake and accumulation of cytotoxic zinc levels. Thus, prostate cancer is a "ZIP1-deficient" malignancy. Testosterone and prolactin are the major hormones that similarly regulate the growth, proliferation, metabolism, and functional activities of the acinar epithelial cells in the peripheral zone (the site of development and progression of malignancy). Testosterone regulation provides the basis for androgen ablation treatment of advanced prostate cancer, which leads to the development of terminal androgen-independent malignancy. Androgen-independent malignancy progresses under the influence of prolactin. These relationships provide the basis for the prevention and treatment of advanced prostate cancer. Clioquinol (zinc ionophore; 5-chloro-7-iodoquinolin-8-ol) is employed to facilitate zinc transport and accumulation in the ZIP1-deficient malignant cells and induce cytotoxic effects. Cabergoline (dopamine agonist) is employed to decrease prolactin production and its role in the progression of androgen-independent malignancy. We propose a clioquinol/cabergoline treatment regimen that will be efficacious for aborting terminal advanced prostate cancer. FDA policies permit this treatment regimen to be employed for these patients.

Prostate specific antigen (PSA) does not provide the reliability that is required for the accurate urology screening of prostate cancer (PCa). Consequently, there has been a major focus and search for a simple, rapid, direct, preferably non-invasive, and highly accurate biomarker and procedure for the urology screening for prostate cancer. Virtually all PCa cases exhibit a marked decrease in zinc in prostate tissue and in prostatic fluid. This is a hallmark "signature" clinical characteristic for all prostate cancers, which provides the clinical basis for zinc screening of PCa. Energy dispersive x-ray fluorescence (EDXRF) of zinc levels in expressed prostatic fluid (EPF) provides > 90% accuracy for the identification of prostate cancer vs normal/benign prostate. An energy dispersive x-ray fluorescence (EDXRF) Zn/Fe ratiometric analysis of expressed prostatic fluid (EPF) can provide > 90% accuracy for the identification of prostate cancer vs normal/benign prostate. This will be achieved by direct EDXRF analysis of a "drop" of EPF directly deposited on a filter paper disc during the urology digital rectal examination of the subject. Interfering and confounding conditions that besiege PSA do not exist in the EDXRF Zn/Fe radiometric analyses. This report reviews the basis for zinc analysis for PCA, provides the supporting evidence that EDXRF Zn/Fe ratiometric analysis of EPF will provide a simple, rapid, direct, non-invasive, and highly accurate biomarker and procedure for the urology screening for prostate cancer.

Hormone-independent malignancy is a major issue of morbidity and deaths that confronts prostate cancer. Despite decades of research, the oncogenic and hormonal implications in the development and progression of prostate malignancy remain mostly speculative. This is largely due to the absence and/or lack of consideration by contemporary clinicians and biomedical investigators regarding the established implications of the co-regulation of testosterone and prolactin in the development, maintenance, metabolism and functions of the prostate gland. Especially relevant is the major metabolic function of production of high levels of citrate by the peripheral zone acinar epithelial cells. Citrate production, along with growth and proliferation by these cells, is regulated by co-existing testosterone and prolactin signaling pathways; and by the oncogenic down-regulation of ZIP1 transporter/zinc/citrate in the development of malignancy. These relationships had not been considered in the issues of hormonedependent malignancy. This review provides the relevant background that has established the dual role of testosterone and prolactin regulation of the prostate gland; which is essential to address the implications in the oncogenic development and progression of hormone-dependent malignancy. The oncogenic factor along with testosterone-dependent and prolactin-dependent relationships leads to the plausible concept that androgen ablation for the treatment of testosteronedependent malignancy results in the development of prolactindependent malignancy; which is testosterone-independent malignancy. Consequently, both testosterone ablation and prolactin ablation are required to prevent and/or abort terminal hormonedependent prostate cancer., Competing Interests: Conflict of interest: the authors declare that there is no potential conflict of interest.

High citrate concentration is a major component in the structure of craniofacial bone, teeth and periodontal tissues of humans and other osteovertebrates. It is now established that citrate incorporation into the apatite/collagen complex of bone is essential for the manifestation of the important biomechanical properties of bone; such as stability, strength, and resistance to fracture. The osteoblasts are specialized citrate-producing cells that provide the citrate incorporated in bone during osteogenic stem cell differentiation for production of new bone; "citration" that occurs in concert with mineralization. Dentin and cementum contain high citrate levels; as contrasted with low citrate in enamel. There exists no information regarding the status and source of incorporated citrate in dentin or in cementum. These are important issues relating to oral, periodontal, craniofacial structures. For example, repair of defects should include new tissue that exhibits the composition, structure, and biomechanical properties of the "normal" tissue; which cannot be achieved in the absence of citrate incorporation in the new tissues. Unfortunately, the presence and role of citrate in these tissues have been largely ignored and unrecognized over the past about 40 years by the dental and medical community. The intent of this review is to re-establish the interest and research regarding the important citrate relationships and issues; with focus on related interests in dentistry.

Reported studies more than forty years ago established that all surgery patients exhibit a marked postoperative hypocitricemia within one day following surgery and persists for seven days and longer. Animals also exhibit the postoperative hypocitricemia. The hypocitricemia results from increased liver clearance of plasma citrate, in which the hepatocytes become capable of transporting and utilizing citrate from plasma. This represents a physiologic/metabolic response during the patient recovery from surgery. The extensive hypocitricemia in response to surgery is not manifested by known citricemic hormones, but is initiated via an unidentified putative endocrine hypocitricemic hormone. In addition to the importance relating to surgery patients, the surgical hypocitricemic effects, along with the liver and hepatic cell effects, will impact virtually all human and animal clinical and experimental studies that include surgical intervention; including the conclusions and translational clinical implications. Unfortunately, the hypocitricemic response to surgery has been ignored for the past forty years, and most contemporary clinicians and biomedical investigators are not aware of this clinical relationship. The intent of this review is to inform members of the medical community of the established hypocitricemic response to surgery and the important role of liver clearance and hepatocyte metabolism of plasma citrate; which, hopefully, will generate interest and research that should be integrated into contemporary issues that involve surgical intervention., Competing Interests: Conflict of interest Author declares that there is no conflict of interest.

Environmental metagenomic analysis is typically accomplished by assigning taxonomy and/or function from whole genome sequencing or 16S amplicon sequences. Both of these approaches are limited, however, by read length, among other technical and biological factors. A nanopore-based sequencing platform, MinION™, produces reads that are ≥1 × 104 bp in length, potentially providing for more precise assignment, thereby alleviating some of the limitations inherent in determining metagenome composition from short reads. We tested the ability of sequence data produced by MinION (R7.3 flow cells) to correctly assign taxonomy in single bacterial species runs and in three types of low-complexity synthetic communities: a mixture of DNA using equal mass from four species, a community with one relatively rare (1%) and three abundant (33% each) components, and a mixture of genomic DNA from 20 bacterial strains of staggered representation. Taxonomic composition of the low-complexity communities was assessed by analyzing the MinION sequence data with three different bioinformatic approaches: Kraken, MG-RAST, and One Codex. Results: Long read sequences generated from libraries prepared from single strains using the version 5 kit and chemistry, run on the original MinION device, yielded as few as 224 to as many as 3497 bidirectional high-quality (2D) reads with an average overall study length of 6000 bp. For the single-strain analyses, assignment of reads to the correct genus by different methods ranged from 53.1% to 99.5%, assignment to the correct species ranged from 23.9% to 99.5%, and the majority of misassigned reads were to closely related organisms. A synthetic metagenome sequenced with the same setup yielded 714 high quality 2D reads of approximately 5500 bp that were up to 98% correctly assigned to the species level. Synthetic metagenome MinION libraries generated using version 6 kit and chemistry yielded from 899 to 3497 2D reads with lengths averaging 5700 bp with up to 98% assignment accuracy at the species level. The observed community proportions for “equal” and “rare” synthetic libraries were close to the known proportions, deviating from 0.1% to 10% across all tests. For a 20-species mock community with staggered contributions, a sequencing run detected all but 3 species (each included at <0.05% of DNA in the total mixture), 91% of reads were assigned to the correct species, 93% of reads were assigned to the correct genus, and >99% of reads were assigned to the correct family. Conclusions: At the current level of output and sequence quality (just under 4 × 103 2D reads for a synthetic metagenome), MinION sequencing followed by Kraken or One Codex analysis has the potential to provide rapid and accurate metagenomic analysis where the consortium is comprised of a limited number of taxa. Important considerations noted in this study included: high sensitivity of the MinION platform to the quality of input DNA, high variability of sequencing results across libraries and flow cells, and relatively small numbers of 2D reads per analysis limit. Together, these limited detection of very rare components of the microbial consortia, and would likely limit the utility of MinION for the sequencing of high-complexity metagenomic communities where thousands of taxa are expected. Furthermore, the limitations of the currently available data analysis tools suggest there is considerable room for improvement in the analytical approaches for the characterization of microbial communities using long reads. Nevertheless, the fact that the accurate taxonomic assignment of high-quality reads generated by MinION is approaching 99.5% and, in most cases, the inferred community structure mirrors the known proportions of a synthetic mixture warrants further exploration of practical application to environmental metagenomics as the platform continues to develop and improve. With further improvement in sequence throughput and error rate reduction, this platform shows great promise for precise real-time analysis of the composition and structure of more complex microbial communities., (© The Author 2017. Published by Oxford University Press.)

Introduction: Efficacious chemotherapy does not exist for treatment or prevention of prostate, liver, and pancreatic carcinomas, and some other cancers that exhibit decreased zinc in malignancy. Zinc treatment offers a potential solution; but its support has been deterred by adverse bias. Areas covered: 1. The clinical and experimental evidence for the common ZIP transporter/Zn down regulation in these cancers. 2. The evidence for a zinc approach to prevent and/or treat these carcinomas. 3. The issues that introduce bias against support for the zinc approach. Expert opinion: ZIP/Zn downregulation is a clinically established common event in prostate, hepatocellular and pancreatic cancers. 2. Compelling evidence supports the plausibility that a zinc treatment regimen will prevent development of malignancy and termination of progressing malignancy in these cancers; and likely other carcinomas that exhibit decreased zinc. 3. Scientifically-unfounded issues that oppose this ZIP/Zn relationship have introduced bias against support for research and funding of a zinc treatment approach. 4. The clinically-established and supporting experimental evidence provide the scientific credibility that should dictate the support for research and funding of a zinc approach for the treatment and possible prevention of these cancers. 5. This is in the best interest of the medical community and the public-at-large., Competing Interests: Declaration of interest The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript.

The human prostate gland contains extremely high zinc levels; which is due to the specialized zinc-accumulating acinar epithelial of the peripheral zone. These cells evolved for their unique capability to produce and secrete extremely levels of citrate, which is achieved by the high cellular zinc level effects on the cell metabolism. This review highlights the specific functional and metabolic alterations that result from the accumulation of the high zinc levels, especially its effects on mitochondrial citrate metabolism and terminal oxidation. The implications of zinc in the development and progression of prostate cancer are described, which is the most consistent hallmark characteristic of prostate cancer. The requirement for decreased zinc resulting from down regulation of ZIP1 to prevent zinc cytotoxicity in the malignant cells is described as an essential early event in prostate oncogenesis. This provides the basis for the concept that an agent (such as the zinc ionophore, clioquinol) that facilitates zinc uptake and accumulation in ZIP1-deficient prostate tumors cells will markedly inhibit tumor growth. In the current absence of an efficacious chemotherapy for advanced prostate cancer, and for prevention of early development of malignancy; a zinc treatment regimen is a plausible approach that should be pursued., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Purpose: Altered zinc levels in malignant cells versus their normal cells have important implications in the development and progression of several cancers. Prostate, pancreatic, and hepatocellular carcinomas exhibit consistent marked zinc decrease in situ in the malignant cells, and other cancers (such as kidney, lung, and thyroid) also exhibit decreased tissue zinc levels. However, zinc levels are increased in breast cancer tissue compared to breast normal tissue, and the contemporary dominant view is that zinc is increased in invasive ductal carcinoma. This has important implications regarding the role and effects of zinc in breast malignancy compared to other cancers, which caused us to initiate this study to either confirm or challenge the contemporary view of an increased zinc level in the invasive ductal malignant cells., Methods: We employed dithizone staining of breast tissue sections and tissue cores to determine the relative in situ cellular zinc levels specifically in the invasive ductal malignant cells as compared to normal ductal epithelium. This approach had not been employed in any of the reported breast studies., Results: The results revealed that the zinc levels are consistently and markedly decreased in the ductal malignant cells as compared with higher prominent zinc levels in the normal ductal epithelium. Decreased zinc is evident in Grade 1 well-differentiated malignancy and in Grade 2 and Grade 3 carcinomas. Among the twenty-five cancer cases in this study, none exhibited increased zinc in the invasive ductal carcinoma compared to the zinc level in the normal ductal epithelium., Conclusions: The decreased zinc levels in breast invasive ductal carcinoma is consistent with prostate, pancreatic, and liver carcinomas in which the decrease in zinc is a required event in the development of malignancy to prevent cytotoxicity that would result from the higher zinc levels in the normal cells. This new understanding requires a redirection in elucidating the mechanisms and factors regarding the regulation of zinc in breast cancer, its potential translational applications as possible biomarkers, and for treatment of breast invasive ductal carcinoma.

The homeostatic maintenance of a normal plasma citrate concentration is an important factor in humans and in animals; and is required for many normal physiological activities. Dysregulation of normal plasma citrate presents pathophysiological hypocitricemic or hypercitricemic conditions. This can lead to clinical consequences in many areas of medicine; such as impaired blood clotting, altered acid/base status, impaired neuromuscular/cardiac activities, hypocitraturia and stone formation, bone disorders with loss of bone strength and increased fractures, hypocitricemia of surgical stress. These important implications of citrate relationships have been largely ignored by the contemporary clinical and biomedical community; to the extent that it is not even described in most current textbooks and review papers. This review describes the physiological, endocrine, and metabolic relationships in the normal regulation and maintenance of plasma citrate; and presents some important clinical consequences of its dysfunctional maintenance. The importance of bone, kidney and liver activities in the maintenance of normal plasma citrate is described along with the citricemic roles of parathyroid hormone, calcitonin and vitamin D. These factors and relationships are presented as the contemporary understanding of the integrated regulation of plasma citrate as the basis for its clinical importance in medicine. The exclusion of these citrate relationships leads to misunderstanding and misrepresentation of physiological and clinical conditions in many issues in medicine and paramedicine areas. The intent of this review is to revive the interest and support for research to address the many unknown and speculative issues of plasma citrate regulation and its important clinical implications. This is in the best interest of the medical community and the public-at-large.

Prostate cancer remains the second leading cause of cancer deaths in males. This is mainly due to the absence of an available efficacious chemotherapy despite decades of research in pursuit of effective treatment approaches. A plausible target for the treatment is the established clinical relationship that the zinc levels in the malignant cells are markedly decreased compared to the normal epithelium in virtually all cases of prostate cancer, and at all stages malignancy. The decrease in zinc results from the downregulation of the functional zinc uptake transporter, ZIP1; which occurs during early development of prostate malignancy. This is an essential requirement for the development of malignancy to prevent the cytotoxic/tumor-suppressor effects of increased zinc on the premalignant and malignant cells. Thus prostate cancer is a ZIP1-deficient malignancy. This relationship provides the basis for a treatment regimen that will facilitate the uptake and accumulation of zinc into the premalignant and malignant cells. In this report we employed a zinc ionophore (clioquinol) approach in the treatment of mice with human ZIP1-deficient prostate tumors (ectopic xenograft model). Clioquinol treatment resulted in 85%inhibition of tumor growth due to the cytotoxic effects of zinc. Coupled with additional results from earlier studies, the compelling evidence provides a plausible approach for the effective treatment of human prostate cancer; including primary site malignancy, hormone-resistant cancer, and metastasis. Additionally, this approach might be effective in preventing the development of malignancy in individuals suspected of presenting with early development of malignancy. Clinical trials are now required in leading to the potential for an efficacious zinc-treatment approach, which is urgently needed for the treatment of prostate cancer.

Salinity is a major driver of bacterial community composition across the globe. Despite growing recognition that different bacterial species are present or active at different salinities, the mechanisms by which salinity structures community composition remain unclear. We tested the hypothesis that these patterns reflect ecological coherence in the salinity preferences of phylogenetic groups using a reciprocal transplant experiment of fresh- and saltwater wetland soils. The salinity of both the origin and host environments affected community composition (16S rRNA gene sequences) and activity (CO2 and CH4 production, and extracellular enzyme activity). These changes in community composition and activity rates were strongly correlated, which suggests the effect of environment on function could be mediated, at least in part, by microbial community composition. Based on their distribution across treatments, each phylotype was categorized as having a salinity preference (freshwater, saltwater, or none) and phylogenetic analyses revealed a significant influence of evolutionary history on these groupings. This finding was corroborated by examining the salinity preferences of high-level taxonomic groups. For instance, we found that the majority of α- and γ-proteobacteria in these wetland soils preferred saltwater, while many β-proteobacteria prefer freshwater. Overall, our results indicate the effect of salinity on bacterial community composition results from phylogenetically-clustered salinity preferences.

Knowledge of the diversity and ecological function of the microbial consortia of James River in Virginia, USA, is essential to developing a more complete understanding of the ecology of this model river system. Metagenomic analysis of James River's planktonic microbial community was performed for the first time using an unamplified genomic library and a 16S rDNA amplicon library prepared and sequenced by Ion PGM and MiSeq, respectively. From the 0.46-Gb WGS library (GenBank:SRR1146621; MG-RAST:4532156.3), 4 × 10(6) reads revealed >3 × 10(6) genes, 240 families of prokaryotes, and 155 families of eukaryotes. From the 0.68-Gb 16S library (GenBank:SRR2124995; MG-RAST:4631271.3; EMB:2184), 4 × 10(6) reads revealed 259 families of eubacteria. Results of the WGS and 16S analyses were highly consistent and indicated that more than half of the bacterial sequences were Proteobacteria, predominantly Comamonadaceae. The most numerous genera in this group were Acidovorax (including iron oxidizers, nitrotolulene degraders, and plant pathogens), which accounted for 10 % of assigned bacterial reads. Polaromonas were another 6 % of all bacterial reads, with many assignments to groups capable of degrading polycyclic aromatic hydrocarbons. Albidiferax (iron reducers) and Variovorax (biodegraders of a variety of natural biogenic compounds as well as anthropogenic contaminants such as polycyclic aromatic hydrocarbons and endocrine disruptors) each accounted for an additional 3 % of bacterial reads. Comparison of these data to other publically-available aquatic metagenomes revealed that this stretch of James River is highly similar to the upper Mississippi River, and that these river systems are more similar to aquaculture and sludge ecosystems than they are to lakes or to a pristine section of the upper Amazon River. Taken together, these analyses exposed previously unknown aspects of microbial biodiversity, documented the ecological responses of microbes to urban effects, and revealed the noteworthy presence of 22 human-pathogenic bacterial genera (e.g., Enterobacteriaceae, pathogenic Pseudomonadaceae, and 'Vibrionales') and 6 pathogenic eukaryotic genera (e.g., Trypanosomatidae and Vahlkampfiidae). This information about pathogen diversity may be used to promote human epidemiological studies, enhance existing water quality monitoring efforts, and increase awareness of the possible health risks associated with recreational use of James River.

Background: Mucinous carcinomas from different organs are morphologically similar and might share similarities at the molecular and biochemical levels that may illuminate their pathogenesis and influence management. The factors involved in the pathogenesis of mucinous carcinomas remain unknown; which is likely one contributor to the current dearth of biomarkers for detection. Because zinc changes are implicated in some cancers e.g., prostate; we assessed the possibility of a similar role in mucinous carcinomas., Methods: The goal of the current work is to study the expression of hZip1 by immunohistochemistry in mucinous carcinomas as compared with non-neoplastic epithelia and conventional carcinomas. Tissue microarray slides containing mucinous carcinomas of the ovary (n = 35), colon (n = 51), stomach (n = 32) and lung (n = 21) were used., Results: hZip1 showed persistent low expression in mucinous compared to ovarian serous carcinomas and normal tissue (P < 0.05), colonic adenocarcinoma and normal mucosa (P < 0.001), and gastric adenocarcinoma and normal epithelium (P < 0.05). hZip1 also showed low expression in pulmonary mucinous carcinomas., Conclusions: hZip1 is consistently decreased in mucinous carcinomas from a variety of organs. Despite the fact that these preliminary findings are unlikely to be of much diagnostic significance, these findings suggest that hZip1 plays a fundamental role in the carcinogenesis of mucinous tumors.

Despite decades of research, no efficacious chemotherapy exists for the treatment of prostate cancer. Malignant prostate zinc levels are markedly decreased in all cases of prostate cancer compared to normal/benign prostate. ZIP1 zinc transporter down regulation decreases zinc to prevent its cytotoxic effects. Thus, prostate cancer is a "ZIP1-deficient" malignancy. A zinc ionophore (e.g. Clioquinol) treatment to increase malignant zinc levels is a plausible treatment of prostate cancer. However, skepticism within the clinical/biomedical research community impedes significant progress leading to such a zinc treatment. This report reviews the clinical and experimental background, and presents new experimental data showing Clioquinol suppression of prostate malignancy; which provides strong support for a zinc ionophore treatment for prostate cancer. Evaluation of often-raised opposing issues is presented. These considerations lead to the conclusion that the compelling evidence dictates that a zinc-treatment approach for prostate cancer should be pursued with additional research leading to clinical trials.

Accurate prediction of denitrification rates remains difficult, potentially owing to complex uncharacterized interactions between resource conditions and denitrifier communities. To better understand how the availability of organic matter (OM) and nitrate (NO3 (-) ), two of the resources most fundamental to denitrifiers, affect these populations and their activity, we performed an in situ resource manipulation in tidal freshwater wetland soils. Soils were augmented with OM to double ambient concentrations, using either compost or plant litter, and fertilized with KNO3 at two levels (low: ∼ 5 mg l(-1)  NO3 (-) -N and high: ∼ 50 mg l(-1)  NO3 (-) -N) in a full factorial design. Community composition of nirS-denitrifers (assessed using terminal restriction fragment length polymorphism) was interactively regulated by both NO3 (-) concentration and OM type, and the associated shifts in community composition were relatively consistent across sampling dates (6, 9 and 12 months of incubation). Denitrification potential (pDNF) rates were also strongly affected by NO3 (-) fertilization and increased by ∼ 10-100-fold. Path analysis revealed that the influence of resource availability on pDNF rates was largely mediated through changes in nirS-denitrifier community composition. These results suggest that a greater understanding of denitrifier community ecology may enable more accurate prediction of denitrification rates., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Background: That citrate is a major indispensible component of bone in humans and in all osteovertebrates has been known for about seventy-five years. Yet, its role and importance in the structure and function of bone and bone formation have remained unknown. However, recent studies have identified that citrate is a major and essential component of the apatite/collagen structure of bone; and that the biomechanical properties of bone (e.g., stability, strength, resistance to fracture) depend on the appropriate incorporation of citrate in the structure of bone. The osteoblasts have recently been identified as citrate-producing cells that provide the citrate that is incorporated in the apatite/collagen structure during osteogenesis. Little is known regarding the factors and mechanisms involved in the regulation of citrate that is incorporated along with mineralization during the process of bone formation. Because of the importance of BMP2 in the initiation of osteogenesis and the development of the osteoblasts, it is essential to determine its possible implication in the development of the citrate-producing capability of the osteoblasts (i.e., "citration") during the formation of mineralized bone nodules., Methods: The goal of this study was to determine if BMP2 promotes the development of citrate-producing osteoblasts for increased citrate incorporation in the formation of mineralized bone nodules. The study employed MC3T3 mesenchyme stem cell osteogenic differentiation in the presence and absence of BMP2., Results: The results showed that BMP2 treatment increased the osteogenic development of mineralized bone nodules. In addition, BMP2 increased osteoblast citrate production and incorporation in the mineralized bone nodule. This was accompanied by increased ZIP1 transporter, which is an essential genetic/metabolic event for citrate-producing cells., Conclusions: The results demonstrate, for the first time, that BMP2 facilitates the osteoblast "citration" process in concert with mineralization during bone formation; and provide confirmation of the important role of osteoblasts as specialized citrate-producing cells in the process of bone formation. However, it is essential to determine if these in vitro effects will occur in vivo in BMP2-implant induction of bone formation. "Citration" is essential for osteoinductive bone to represent the chemical, structural, and biomechanical properties of "normal" bone.