ABSTRACT

Aspergillus flavus, a ubiquitous and saprophytic fungus, is the second most common cause of aspergillosis worldwide. Several mechanisms contribute to the establishment of the fungal infection. Extracellular vesicles (EVs) have been described as "virulence factor delivery bags" in several fungal species, demonstrating a crucial role during the infection. In this study, we evaluated production of A. flavus EVs and their immunomodulatory functions. We verified that A. flavus EVs induce macrophages to produce inflammatory mediators, such as nitric oxide, tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-1β. Furthermore, the A. flavus EVs enhance phagocytosis and killing by macrophages and induce M1 macrophage polarization in vitro. In addition, a prior inoculation of A. flavus EVs in Galleria mellonella larvae resulted in a protective effect against the fungal infection. Our findings suggest that A. flavus EVs are biologically active and affect the interaction between A. flavus and host immune cells, priming the innate immune system to eliminate the fungal infection. Collectively, our results suggest that A. flavus EVs play a crucial role in aspergillosis.

IMPORTANCE Immunocompromised patients are susceptible to several fungal infections. The genus Aspergillus can cause increased morbidity and mortality. Developing new therapies is essential to understand the fungal biology mechanisms. Fungal EVs carry important virulence factors, thus playing pivotal roles in fungal pathophysiology. No study to date has reported EV production by Aspergillus flavus, a fungus considered to be the second most common cause of aspergillosis and relevant food contaminator found worldwide. In this study, we produced A. flavus EVs and evaluated the in vitro immunomodulatory effects of EVs on bone marrow-derived macrophages (BMDMs) and in vivo effects in a Galleria mellonella model.

ABSTRACT

In numerous instances, tracking the biological significance of a nucleic acid sequence can be augmented through the identification of environmental niches in which the sequence of interest is present. Many metagenomic data sets are now available, with deep sequencing of samples from diverse biological niches. While any individual metagenomic data set can be readily queried using web-based tools, meta-searches through all such data sets are less accessible. In this brief communication, we demonstrate such a meta-metagenomic approach, examining close matches to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in all high-throughput sequencing data sets in the NCBI Sequence Read Archive accessible with the "virome" keyword. In addition to the homology to bat coronaviruses observed in descriptions of the SARS-CoV-2 sequence (F. Wu, S. Zhao, B. Yu, Y. M. Chen, et al., Nature 579:265–269, 2020, https://doi.org/10.1038/s41586-020-2008-3; P. Zhou, X. L. Yang, X. G. Wang, B. Hu, et al., Nature 579:270–273, 2020, https://doi.org/10.1038/s41586-020-2012-7), we note a strong homology to numerous sequence reads in metavirome data sets generated from the lungs of deceased pangolins reported by Liu et al. (P. Liu, W. Chen, and J. P. Chen, Viruses 11:979, 2019, https://doi.org/10.3390/v11110979). While analysis of these reads indicates the presence of a similar viral sequence in pangolin lung, the similarity is not sufficient to either confirm or rule out a role for pangolins as an intermediate host in the recent emergence of SARS-CoV-2. In addition to the implications for SARS-CoV-2 emergence, this study illustrates the utility and limitations of meta-metagenomic search tools in effective and rapid characterization of potentially significant nucleic acid sequences.

IMPORTANCE Meta-metagenomic searches allow for high-speed, low-cost identification of potentially significant biological niches for sequences of interest.

Molar element ratio analysis of element concentrations consists of four basic tools that provide substantial insight into the lithogeochemistry (and mineralogy) of rocks under examination. These tools consist of: (1) conserved element ratio analysis; (2) Pearce element ratio analysis; (3) general element ratio analysis; and (4) lithogeochemical mineral mode analysis. Conserved element ratio analysis is useful in creating a chemostratigraphic model for the host rocks to mineral deposits, whereas Pearce element ratio analysis and general element ratio analysis are primarily used to identify mineralogical and metasomatic controls on rock compositions and to investigate and quantify the extent of the material transfers that formed the host rocks and mineralization. Lithogeochemical mineral mode analysis converts element concentrations into mineral concentrations using a matrix-based change-of-basis operation, allowing lithogeochemical data to be interpreted in terms of mineral modes. It can be used to provide proper names to rocks, an important activity for an exploration geologist because of the implications that rock names have on genetic processes and mineral deposit models.

This paper provides a review of the theoretical foundations of each of these four tools and then illustrates how these techniques have been used in a variety of exploration applications to assist in the search for, evaluation and planning of, and the mining of mineral deposits. Examples include the evaluation of total digestion lithogeochemical datasets from mineral deposits hosted by igneous and sedimentary rocks and formed by hydrothermal and igneous processes. In addition, this paper illustrates a more recent geometallurgical application of these methods, whereby the mineral proportions determined by lithogeochemical mineral mode analysis are used to predict rock properties and obtain the ore body knowledge critical for resource evaluation, mine planning, mining and mine remediation.

Thematic collection: This article is part of the Exploration 17 collection available at: https://www.lyellcollection.org/cc/exploration-17

Among the emerging techniques to detect the real footprint of buried ore deposits is isotope tracing. Novel and automated preparation systems such as continuous flow isotope ratio mass spectrometry, off-axis integrated cavity output spectroscopy for isotopic compositions of selected molecules, multi-collector inductively coupled-plasma mass spectrometry (ICP-MS), triple quadrupole ICP-MS, laser ablation ICP-MS, and a multitude of inline preparation systems have facilitated the use of isotopes as tracers in mineral exploration, as costs for isotope analyses have decreased and the time required for the analyses has improved. In addition, the isotope systems being used have expanded beyond the traditional light stable and Pb isotopes to include a multitude of elements that behave differently during processes that promote the mobilization of elements during both primary and secondary dispersion. Isotopes are also being used to understand barren areas that lack a critical process to form an ore deposit and to reveal precise redox mechanisms. The goal is to be able to use isotopes to reflect a definitive process that occurs in association with the deposit and not in barren systems, and then to relate these to something that is easier to measure, namely elemental concentrations. As new generations of exploration and environmental scientists are becoming more comfortable with the application of isotopes to effectively trace processes involved in geoscience, and new technologies for rapid and inexpensive analyses of isotopes are continually being developed, novel applications of isotope tracing are becoming more mainstream.

Thematic collection: This article is part of the Exploration 17 collection available at: https://www.lyellcollection.org/cc/exploration-17

There is a pressing need for new exploration tools to target and vector towards mineralization in covered terrains. Groundwater provides a valuable and under-utilized geochemical sampling medium, and represents an important and cost-effective tool to expose covered terrains to systematic exploration. For Au exploration, researchers agree the best hydrogeochemistry pathfinder is dissolved Au itself, with additional potential from other pathfinders (albeit non-unique) such as As, Ag, W and Mo. Despite Au's relatively low solubility, with rigorous field protocols and appropriate analytical methods, explorers can respond to dissolved Au directly with robust parts per trillion (ppt)-level analyses.

Even with ppt-level analyses, a practical implication of Au's low solubility is that a deposit's dissolved Au signature is generally weaker than seen in other more mobile pathfinders, producing a smaller detectable footprint, which must be considered when designing exploration programmes. Using purpose-drilled groundwater sampling bores, explorers can collect groundwater samples at the density required to respond to dissolved Au where existing borehole coverage is otherwise insufficient. In addition to its use at the regional scale, with even tighter sample density, hydrogeochemistry also shows promise at the project scale, allowing the 3D modelling of pathfinder dispersion.

For hydrogeochemistry to be widely adopted for Au exploration, explorers need confidence in ppt-level dissolved Au analyses, and the context to understand their significance. This paper aims to address these topics and provide a straightforward starting point for Au explorers interested in applying hydrogeochemistry by: (i) summarizing examples of regional sampling programmes and more focused case studies to illustrate how covered Au deposits create measurable dissolved Au footprints distinguishable from background; and (ii) sharing examples of dissolved Au analyses that are being integrated into exploration at the regional and project scales.

As seen in the results, the distributions of dissolved Au in the regional- and project-scale programmes show remarkably similar and easy to interpret high-contrast, low-frequency anomalies against relatively low backgrounds. These are desirable attributes of any geochemical pathfinder. When combined with the benefits of hydrogeochemistry v. other geochemical exploration tools (e.g. groundwater can create larger footprints requiring fewer samples to detect, and groundwater can recharge from depth to reflect deeper mineralization), dissolved Au is a powerful pathfinder ideally suited for Au exploration in covered terrains.

While this paper focuses on the use of dissolved Au, additional pathfinders can provide valuable information, including indications of lithological changes, hydrothermal alteration and different styles of mineralization, as well as opportunities to use secondary pathfinders when sample density or local conditions may not result in detectable dissolved Au signatures.

Thematic collection: This article is part of the Exploration 17 collection available at: https://www.lyellcollection.org/cc/exploration-17

Capture bioprocessing unit operations were previously shown to clear or kill several log₁₀ of a model mycoplasma Acholeplasma laidlawii in lab-scale spike/removal studies. Here, we confirm this observation with two additional mollicute species relevant to biotechnology products for human use: Mycoplasma orale and Mycoplasma arginini. Clearance of M. orale and M. arginini from protein A column purification was similar to that seen with A. laidlawii, though some between cycle carryover was evident, especially for M. orale. However, on-resin growth studies for all three species revealed that residual mycoplasma in a column slowly die off over time rather than expanding further. Solvent/detergent exposure completely inactivated M. arginini though detectable levels of M. orale remained. A small-scale model of a commercial low-pH hold step did inactivate live M. orale, but this inactivation required a lower pH set point and occurred with slower kinetics than previously seen with A. laidlawii. Additionally, ultraviolet-C irradiation was shown to be effective for A. laidlawii and M. orale inactivation whereas virus-retentive filters for upstream and downstream processes, as expected, cleared A. laidlawii. These data argue that M. orale and M. arginini overall would be largely cleared by early bioprocessing steps as shown previously for A. laidlawii, and that barrier technologies can effectively reduce the risk from media components. For some unit operations, M. orale and M. arginini may be hardier, and require more stringent processing or equipment cleaning conditions to assure effective mycoplasma reduction. By exploring how some of the failure modes in commercial antibody manufacturing processes can still eliminate mycoplasma burden, we demonstrate that required best practices assure biotechnology products will be safe for patients.

A vial-capping process for lyophilization stopper configurations was previously quantified using residual seal force (RSF). A correlation between RSF and container closure integrity (CCI) was established, and component positional offsets were identified to be the primary source of variability in RSF measurements.

To gain insight into the effects of stopper geometry on CCI, serum stoppers with the same rubber formulation were investigated in this study. Unlike lyophilization stoppers that passed CCI (per helium leak testing) even with RSF of 0 N owing to their excellent valve seal, serum stoppers consistently failed CCI when RSF was <15.8 N. When the plug was removed, both types of stoppers exhibited a comparable critical lower RSF limit (19–20 N), below which CCI could not be maintained. When CCI was retested at later time points (up to 6 mo), some previously failed vials passed CCI, suggesting that CCI improvement might be related to rubber relaxation (viscous flow), which can fill minor imperfections on the vial finish.

To confirm component positional offsets are the primary sources of RSF variability, a novel quantification tool—micro-computed tomography (micro-CT)—was used in this study. Micro-CT provided images for quantification of positional offsets of the cap and stopper that directly correlated with RSF fluctuations. Serum stoppers and lyophilization stoppers are comparable in RSF variations, although lyophilization stoppers are more robust in CCI. The use of micro-CT provides a nondestructive and innovative tool in quantitatively analyzing component features of capped vials that would otherwise be difficult to investigate.

Viruses and mobile genetic elements are molecular parasites or symbionts that coevolve with nearly all forms of cellular life. The route of virus replication and protein expression is determined by the viral genome type. Comparison of these routes led to the classification of viruses into seven "Baltimore classes" (BCs) that define the major features of virus reproduction. However, recent phylogenomic studies identified multiple evolutionary connections among viruses within each of the BCs as well as between different classes. Due to the modular organization of virus genomes, these relationships defy simple representation as lines of descent but rather form complex networks. Phylogenetic analyses of virus hallmark genes combined with analyses of gene-sharing networks show that replication modules of five BCs (three classes of RNA viruses and two classes of reverse-transcribing viruses) evolved from a common ancestor that encoded an RNA-directed RNA polymerase or a reverse transcriptase. Bona fide viruses evolved from this ancestor on multiple, independent occasions via the recruitment of distinct cellular proteins as capsid subunits and other structural components of virions. The single-stranded DNA (ssDNA) viruses are a polyphyletic class, with different groups evolving by recombination between rolling-circle-replicating plasmids, which contributed the replication protein, and positive-sense RNA viruses, which contributed the capsid protein. The double-stranded DNA (dsDNA) viruses are distributed among several large monophyletic groups and arose via the combination of distinct structural modules with equally diverse replication modules. Phylogenomic analyses reveal the finer structure of evolutionary connections among RNA viruses and reverse-transcribing viruses, ssDNA viruses, and large subsets of dsDNA viruses. Taken together, these analyses allow us to outline the global organization of the virus world. Here, we describe the key aspects of this organization and propose a comprehensive hierarchical taxonomy of viruses.

Although enteroviruses are associated with a wide variety of diseases and conditions, their mode of replication is well conserved. Their genome is carried as a single, positive-sense RNA strand. At the 5' end of the strand is an approximately 90-nucleotide self-complementary region called the 5' cloverleaf, or the oriL. This noncoding region serves as a platform upon which host and virus proteins, including the 3B, 3C, and 3D virus proteins, assemble in order to initiate replication of a negative-sense RNA strand. The negative strand in turn serves as a template for synthesis of multiple positive-sense RNA strands. Building on structural studies of individual RNA stem-loops, the structure of the intact 5' cloverleaf from rhinovirus has recently been determined via nuclear magnetic resonance/small-angle X-ray scattering (NMR/SAXS)-based methods, while structures have also been determined for enterovirus 3A, 3B, 3C, and 3D proteins. Analysis of these structures, together with structural and modeling studies of interactions between host and virus proteins and RNA, has begun to provide insight into the enterovirus replication mechanism and the potential to inhibit replication by blocking these interactions.

In bacterial populations, quorum sensing (QS) systems participate in the regulation of specialization processes and regulate collective behaviors that mediate interactions and allow survival of the species. In Gram-positive bacteria, QS systems of the RRNPP family (Rgg, Rap, NprR, PlcR, and PrgX) consist of intracellular receptors and their cognate signaling peptides. Two of these receptors, Rap and NprR, have regained attention in Bacillus subtilis and the Bacillus cereus group. Some Rap proteins, such as RapH and Rap60, are multifunctional and/or redundant in function, linking the specialization processes of sporulation and competence, as well as global expression changes in the transition phase in B. subtilis. NprR, an evolutionary intermediate between Rap and RRNPP transcriptional activators, is a bifunctional regulator that modulates sporulation initiation and activates nutrient scavenging genes. In this review, we discuss how these receptors switch between functions and connect distinct signaling pathways. Based on structural evidence, we propose that RapH and Rap60 should be considered moonlighting proteins. Additionally, we analyze an evolutionary and ecological perspective to understand the multifunctionality and functional redundancy of these regulators in both Bacillus spp. and non-Bacillus Firmicutes. Understanding the mechanistic, structural, ecological, and evolutionary basis for the multifunctionality and redundancy of these QS systems is a key step for achieving the development of innovative technologies for health and agriculture.

Streptococcus pyogenes (Lancefield group A Streptococcus [GAS]) is a β-hemolytic human-selective pathogen that is responsible for a large number of morbid and mortal infections in humans. For efficient infection, GAS requires different types of surface proteins that provide various mechanisms for evading human innate immune responses, thus enhancing pathogenicity of the bacteria. Many such virulence-promoting proteins, including the major surface signature M protein, are translocated after biosynthesis through the cytoplasmic membrane and temporarily tethered to this membrane via a type 1 transmembrane domain (TMD) positioned near the COOH terminus. In these proteins, a sorting signal, LPXTG, is positioned immediately upstream of the TMD, which is cleaved by the membrane-associated transpeptidase, sortase A (SrtA), leading to the covalent anchoring of these proteins to newly emerging l-Ala–l-Ala cross-bridges of the growing peptidoglycan cell wall. Herein, we show that inactivation of the srtA gene in a skin-tropic pattern D GAS strain (AP53) results in retention of the M protein in the cell membrane. However, while the isogenic AP53 srtA strain is attenuated in overall pathogenic properties due to effects on the integrity of the cell membrane, our data show that the M protein nonetheless can extend from the cytoplasmic membrane through the cell wall and then to the surface of the bacteria and thereby retain its important properties of productively binding and activating fluid-phase host plasminogen (hPg). The studies presented herein demonstrate an underappreciated additional mechanism of cell surface display of bacterial virulence proteins via their retention in the cell membrane and extension to the GAS surface.

IMPORTANCE Group A Streptococcus pyogenes (GAS) is a human-specific pathogen that produces many surface factors, including its signature M protein, that contribute to its pathogenicity. M proteins undergo specific membrane localization and anchoring to the cell wall via the transpeptidase sortase A. Herein, we explored the role of sortase A function on M protein localization, architecture, and function, employing, a skin-tropic GAS isolate, AP53, which expresses a human plasminogen (hPg)-binding M (PAM) Protein. We showed that PAM anchored in the cell membrane, due to the targeted inactivation of sortase A, was nonetheless exposed on the cell surface and functionally interacted with host hPg. We demonstrate that M proteins, and possibly other sortase A-processed proteins that are retained in the cell membrane, can still function to initiate pathogenic processes by this underappreciated mechanism.

The type VI secretion system (T6SS) is a weapon for delivering effectors into target cells that is widespread in Gram-negative bacteria. The T6SS is a highly versatile machine, as it can target both eukaryotic and prokaryotic cells, and it has been proposed that T6SSs are adapted to the specific needs of each bacterium. The expression of T6SS gene clusters and the activation of the secretion apparatus are therefore tightly controlled. In enteroaggregative Escherichia coli (EAEC), the sci1 T6SS gene cluster is subject to a complex regulation involving both the ferric uptake regulator (Fur) and DNA adenine methylase (Dam)-dependent DNA methylation. In this study, an additional, internal, promoter was identified within the sci1 gene cluster using +1 transcriptional mapping. Further analyses demonstrated that this internal promoter is controlled by a mechanism strictly identical to that of the main promoter. The Fur binding box overlaps the –10 transcriptional element and a Dam methylation site, GATC-32. Hence, the expression of the distal sci1 genes is repressed and the GATC-32 site is protected from methylation in iron-rich conditions. The Fur-dependent protection of GATC-32 was confirmed by an in vitro methylation assay. In addition, the methylation of GATC-32 negatively impacted Fur binding. The expression of the sci1 internal promoter is therefore controlled by iron availability through Fur regulation, whereas Dam-dependent methylation maintains a stable ON expression in iron-limited conditions.

IMPORTANCE Bacteria use weapons to deliver effectors into target cells. One of these weapons, the type VI secretion system (T6SS), assembles a contractile tail acting as a spring to propel a toxin-loaded needle. Its expression and activation therefore need to be tightly regulated. Here, we identified an internal promoter within the sci1 T6SS gene cluster in enteroaggregative E. coli. We show that this internal promoter is controlled by Fur and Dam-dependent methylation. We further demonstrate that Fur and Dam compete at the –10 transcriptional element to finely tune the expression of T6SS genes. We propose that this elegant regulatory mechanism allows the optimum production of the T6SS in conditions where enteroaggregative E. coli encounters competing species.

An aberrant bi-apical Follicucullus specimen (Albaillellaria, Radiolaria) has been discovered from an upper Guadalupian (Middle Permian) chert block of the Kamiaso Unit of the Mino terrane, central Japan. If this specimen was formed with double malformation, it would be the oldest record of this phenomenon in radiolarians and the first record of its kind in Albaillellaria.

The Henry Buckley Collection of Planktonic Foraminifera at the Natural History Museum in London (NHMUK) consists of 1665 single-taxon slides housing 23 897 individuals from 203 sites in all the major ocean basins, as well as a vast research library of Scanning Electron Microscope (SEM) photomicrographs. Buckley picked the material from the NHMUK Ocean-Bottom Deposit Collection and also from fresh tow samples. However, his collection remains largely unused as he was discouraged by his managers in the Mineralogy Department from working on or publicizing the collection. Nevertheless, Buckley published pioneering papers on isotopic interpretation of oceanographic and climatic change and was one of the first workers to investigate foraminiferal wall structure using the SEM technique. Details of the collection and images of each slide are available via the NHMUK Data Portal (http://dx.doi.org/10.5519/0035055). The Buckley Collection and its associated Ocean-Bottom Deposit Collection have great potential for taxon-specific studies as well as geochemical work, and both collections are available on request.

A new larger benthic porcelaneous foraminifer of soritid affinity is described as Tarburina zagrosiana n. gen., n. sp. from the late Maastrichtian of the Tarbur Formation, Zagros Zone, SW Iran. It occurs in foraminiferal–dasycladalean wackestones and packstones, in association with Loftusia ssp., dicyclinids/cuneolinids, Neobalkhania bignoti Cherchi & Schroeder, Gyroconulina columellifera Schroeder & Darmoian, Spirolina? farsiana Schlagintweit & Rashidi, Broeckina cf. dufrenoyi (d'Archiac), other benthic foraminifers, and dasycladalean algae. Due to its elongate test and marginal chamber subdivision by aligned vertical partitions, Tarburina n. gen. can be compared with representatives of the Praerhapydionininae. The interio-marginal slit-like foramina/aperture of Tarburina represents an outstanding feature in complex porcelaneous taxa. The monospecific genus Tarburina is considered a Maastrichtian newcomer within the Late Cretaceous Global Community Maturation cycle of larger benthic foraminifera. A biostratigraphic and palaeobiogeographical restriction seems possible, as reported for many other Late Cretaceous larger benthic foraminifera.

An exceptionally well-preserved specimen of the articulated rhodophyte Permocalculus, compared with P. tenellus sensu Elliott, 1955, is described from fine-grained Upper Permian limestones of the Khuff Formation of Saudi Arabia. Longitudinal medullary and sheaf-like cortical filaments extend through the uniserial series of elongate-globular, concave- and convex-terminating, interlocking segments for which they are interpreted to have functioned in articulation. The filaments tend to splay and branch laterally into the cortex where they terminate at the pores. At the terminal aperture, the filaments extend as bifurcating and possibly trifurcating branches and may serve as the origin of a new segment. Numerous elongate-globular chambers, up to five in each row and intimately involved with the filaments, are developed in the outer medulla and are considered to represent reproductive sporangia. The specimen is considered to have occupied predominantly low-energy, normal to slightly elevated salinity, shallow conditions within the subtidal regime of a lagoon.

New scanning electron microscope observations of unadulterated calcareous nannofossil assemblages on lamina surfaces of Cretaceous Tanzania Drilling Project sediments reveal high diversity in the <3 µm size-range and high abundances of small and frangible morphologies. These assemblages prompt comparison to modern assemblages, which show similar high diversity and abundance of very small and fragile taxa, although these assemblages are generally not preserved in the fossil record due to taphonomic filtering. Not only are there broad similarities between the general composition of modern assemblages and those of the Tanzanian lagerstätte, but also our discovery of several new Cretaceous taxa provides evidence for greatly extended fossil lineages of extant orders, with implications for both deep-time biodiversity divergence and survival through the end-Cretaceous mass extinction. Our findings include: new species that are the first-recorded Mesozoic representatives of the extant Syracosphaeraceae and Papposphaeraceae; potentially previously unrecorded diversity in the Mesozoic Calciosoleniaceae, another extant order, represented by extant species that have been described already; and new species and unusually high abundances of the Mesozoic Stephanolithiaceae. We also highlight the extended range of an incertae sedis Cenozoic genus, Ellipsolithus, into at least the Turonian.

Here, we describe seven new miniscule to very small Cretaceous species: Syracosphaera antiqua, S. repagula, Pocillithus macleodii, P. crucifer, Stradnerlithus wendleri, S.? haynesiae and Tortolithus foramen.

ABSTRACTBackground:The focused assessment with sonography for HIV-associated tuberculosis (TB) (FASH) ultrasound protocol has been increasingly used to help clinicians diagnose TB. We sought to quantify the diagnostic utility of FASH for TB among individuals with HIV in Malawi.Methods:Between March 2016 and August 2017, 210 adults with HIV who had 2 or more signs and symptoms that were concerning for TB (fever, cough, night sweats, weight loss) were enrolled from a public HIV clinic in Lilongwe, Malawi. The treating clinicians conducted a history, physical exam, FASH protocol, and additional TB evaluation (laboratory diagnostics and chest radiography) on all participants. The clinician made a final treatment decision based on all available information. At the 6-month follow-up visit, we categorized participants based on clinical outcomes and diagnostic tests as having probable/confirmed TB or unlikely TB; association of FASH with probable/confirmed TB was calculated using Fisher's exact tests. The impact of FASH on empiric TB treatment was determined by asking the clinicians prospectively about whether they would start treatment at 2 time points in the baseline visit: (1) after the initial history and physical exam; and (2) after history, physical exam, and FASH protocol.Results:A total of 181 participants underwent final analysis, of whom 56 were categorized as probable/confirmed TB and 125 were categorized as unlikely TB. The FASH protocol was positive in 71% (40/56) of participants with probable/confirmed TB compared to 24% (30/125) of participants with unlikely TB (odds ratio=7.9, 95% confidence interval=3.9,16.1; P<.001). Among those classified as confirmed/probable TB, FASH increased the likelihood of empiric TB treatment before obtaining any other diagnostic studies from 9% (5/56) to 46% (26/56) at the point-of-care. For those classified as unlikely TB, FASH increased the likelihood of empiric treatment from 2% to 4%.Conclusion:In the setting of HIV coinfection in Malawi, FASH can be a helpful tool that augments the clinician's ability to make a timely diagnosis of TB.

BACKGROUND

Continuous glucose monitoring (CGM) provides important information to aid in achieving glycemic targets in people with diabetes.

OBJECTIVE

Sodium–glucose cotransporter 2 (SGLT2) inhibition causes an increase in endogenous glucose production (EGP). However, the mechanisms are unclear. We studied the effect of SGLT2 inhibitors on EGP in subjects with type 2 diabetes (T2D) and without diabetes (non-DM) in kidney transplant recipients with renal denervation.

OBJECTIVE

With rising health care costs and finite health care resources, understanding the population needs of different type 2 diabetes mellitus (T2DM) patient subgroups is important. Sparse data exist for the application of population segmentation on health care needs among Asian T2DM patients. We aimed to segment T2DM patients into distinct classes and evaluate their differential health care use, diabetes-related complications, and mortality patterns.

OBJECTIVE

Type 2 diabetes is characterized by increased death rate. In order to tackle this dramatic event, it becomes essential to discover novel biomarkers capable of identifying high-risk patients to be exposed to more aggressive preventive and treatment strategies. hs-CRP and serum amyloid P component (SAP) are two acute-phase inflammation proteins, which interact physically and share structural and functional features. We investigated their combined role in associating with and improving prediction of mortality in type 2 diabetes.

OBJECTIVE

We investigated the association between changes in weight status from childhood through adulthood and subsequent type 2 diabetes risks and whether educational attainment, smoking, and leisure time physical activity (LTPA) modify this association.

In patients with cancer-associated venous thromboembolism, knowledge of the estimated rate of recurrent events is important for clinical decision-making regarding anticoagulant therapy. The Ottawa score is a clinical prediction rule designed for this purpose, stratifying patients according to their risk of recurrent venous thromboembolism during the first six months of anticoagulation. We conducted a systematic review and meta-analysis of studies validating either the Ottawa score in its original or modified versions. Two investigators independently reviewed the relevant articles published from 1st June 2012 to 15th December 2018 and indexed in MEDLINE and EMBASE. Nine eligible studies were identified; these included a total of 14,963 patients. The original score classified 49.3% of the patients as high-risk, with a sensitivity of 0.7 [95% confidence interval (CI): 0.6-0.8], a 6-month pooled rate of recurrent venous thromboembolism of 18.6% (95%CI: 13.9-23.9). In the low-risk group, the recurrence rate was 7.4% (95%CI: 3.4-12.5). The modified score classified 19.8% of the patients as low-risk, with a sensitivity of 0.9 (95%CI: 0.4-1.0) and a 6-month pooled rate of recurrent venous thromboembolism of 2.2% (95%CI: 1.6-2.9). In the high-risk group, recurrence rate was 10.2% (95%CI: 6.4-14.6). Limitations of our analysis included type and dosing of anticoagulant therapy. We conclude that new therapeutic strategies are needed in patients at high risk for recurrent cancer-associated venous thromboembolism. Low-risk patients, as per the modified score, could be good candidates for oral anticoagulation. (This systematic review was registered with the International Prospective Registry of Systematic Reviews as: PROSPERO CRD42018099506).

MYC translocations, a hallmark of Burkitt lymphoma, occur in 5-15% of diffuse large B-cell lymphoma, and have a negative prognostic impact. Numerical aberrations of MYC have also been detected in these patients, but their incidence and prognostic role are still controversial. We analyzed the clinical impact of MYC increased copy number on 385 patients with diffuse large B-cell lymphoma screened at diagnosis for MYC, BCL2, and BCL6 rearrangements. We enumerated the number of MYC copies, defining as amplified those cases with an uncountable number of extra-copies. The prevalence of MYC translocation, increased copy number and amplification was 8.8%, 15%, and 1%, respectively. Patients with 3 or 4 gene copies, accounting for more than 60% of patients with MYC copy number changes, had a more favorable outcome compared to patients with >4 copies or translocation of MYC, and were not influenced by the type of treatment received as first-line. Stratification according to the number of MYC extra-copies showed a negative correlation between an increasing number of copies and survival. Patients with >7 copies or the amplification of MYC had the poorest prognosis. Patients with >4 copies of MYC showed a similar, trending towards worse prognosis compared to patients with MYC translocation. The survival of patients with >4 copies, translocation or amplification of MYC seemed to be superior if intensive treatments were used. Our study underlines the importance of fluorescence in situ hybridization testing at diagnosis of diffuse large B-cell lymphoma to detect the rather frequent and clinically significant numerical aberrations of MYC.

B-cell receptor (BCR) signaling pathway components represent promising treatment targets in multiple B-cell malignancies including diffuse large B-cell lymphoma (DLBCL). In in vitro and in vivo model systems, a subset of DLBCLs depend upon BCR survival signals and respond to proximal BCR/phosphoinositide 3 kinase (PI3K) blockade. However, single-agent BCR pathway inhibitors have had more limited activity in patients with DLBCL, underscoring the need for indicators of sensitivity to BCR blockade and insights into potential resistance mechanisms. Here, we report highly significant transcriptional upregulation of C-X-C chemokine receptor 4 (CXCR4) in BCR-dependent DLBCL cell lines and primary tumors following chemical spleen tyrosine kinase (SYK) inhibition, molecular SYK depletion or chemical PI3K blockade. SYK or PI3K inhibition also selectively upregulated cell surface CXCR4 protein expression in BCR-dependent DLBCLs. CXCR4 expression was directly modulated by fork-head box O1 via the PI3K/protein kinase B/forkhead box O1 signaling axis. Following chemical SYK inhibition, all BCR-dependent DLBCLs exhibited significantly increased stromal cell-derived factor-1α (SDF-1α) induced chemotaxis, consistent with the role of CXCR4 signaling in B-cell migration. Select PI3K isoform inhibitors also augmented SDF-1α induced chemotaxis. These data define CXCR4 upregulation as an indicator of sensitivity to BCR/PI3K blockade and identify CXCR4 signaling as a potential resistance mechanism in BCR-dependent DLBCLs.

Mutation and translocation of fibroblast growth factor receptors often lead to aberrant signaling and cancer. This work focuses on the t(8;22)(p11;q11) chromosomal translocation which creates the breakpoint cluster region (BCR) fibroblast growth factor receptor1 (FGFR1) (BCR-FGFR1) fusion protein. This fusion occurs in stem cell leukemia/lymphoma, which can progress to atypical chronic myeloid leukemia, acute myeloid leukemia, or B-cell lymphoma. This work focuses on the biochemical characterization of BCR-FGFR1 and identification of novel therapeutic targets. The tyrosine kinase activity of FGFR1 is required for biological activity as shown using transformation assays, interleukin-3 independent cell proliferation, and liquid chromatography/mass spectroscopy analyses. Furthermore, BCR contributes a coiled-coil oligomerization domain, also essential for oncogenic transformation by BCR-FGFR1. The importance of salt bridge formation within the coiled-coil domain is demonstrated, as disruption of three salt bridges abrogates cellular transforming ability. Lastly, BCR-FGFR1 acts as a client of the chaperonin heat shock protein 90 (Hsp90), suggesting that BCR-FGFR1 relies on Hsp90 complex to evade proteasomal degradation. Transformed cells expressing BCR-FGFR1 are sensitive to the Hsp90 inhibitor Ganetespib, and also respond to combined treatment with Ganetespib plus the FGFR inhibitor BGJ398. Collectively, these data suggest novel therapeutic approaches for future stem cell leukemia/lymphoma treatment: inhibition of BCR oligomerization by disruption of required salt bridges; and inhibition of the chaperonin Hsp90 complex.

As an alternative to lifelong insulin supplementation, potentiation of immune tolerance in patients with type 1 diabetes could prevent the autoimmune destruction of pancreatic islet β-cells. This study was aimed to assess whether the G3c monoclonal antibody (mAb), which triggers the glucocorticoid-induced TNFR-related (Gitr) costimulatory receptor, promotes the expansion of regulatory T cells (Tregs) in SV129 (wild-type) and diabetic-prone NOD mice. The delivery of the G3c mAb via G3C hybridoma cells enveloped in alginate-based microcapsules (G3C/cps) for 3 weeks induced Foxp3⁺ Treg-cell expansion in the spleen of wild-type mice but not in Gitr^–/– mice. G3C/cps also induced the expansion of nonconventional Cd4⁺Cd25^–/lowFoxp3^lowGitr^int/high (GITR single-positive [sp]) Tregs. Both Cd4⁺Cd25⁺Gitr^highFoxp3⁺ and GITRsp Tregs (including also antigen-specific cells) were expanded in the spleen and pancreas of G3C/cps-treated NOD mice, and the number of intact islets was higher in G3C/cps-treated than in empty cps-treated and untreated animals. Consequently, all but two G3C/cps-treated mice did not develop diabetes and all but one survived until the end of the 24-week study. In conclusion, long-term Gitr triggering induces Treg expansion, thereby delaying/preventing diabetes development in NOD mice. This therapeutic approach may have promising clinical potential for the treatment of inflammatory and autoimmune diseases.

Accumulation of lipid in skeletal muscle is thought to be related to the development of insulin resistance and type 2 diabetes. Initial work in this area focused on accumulation of intramuscular triglyceride; however, bioactive lipids such as diacylglycerols and sphingolipids are now thought to play an important role. Specific species of these lipids appear to be more negative toward insulin sensitivity than others. Adding another layer of complexity, localization of lipids within the cell appears to influence the relationship between these lipids and insulin sensitivity. This article summarizes how accumulation of total lipids, specific lipid species, and localization of lipids influence insulin sensitivity in humans. We then focus on how these aspects of muscle lipids are impacted by acute and chronic aerobic and resistance exercise training. By understanding how exercise alters specific species and localization of lipids, it may be possible to uncover specific lipids that most heavily impact insulin sensitivity.

Yuta Sakae, Akira Oikawa, Yuki Sugiura, Masatoshi Mita, Shuhei Nakamura, Toshiya Nishimura, Makoto Suematsu, and Minoru Tanaka

The teleost fish, medaka (Oryzias latipes), employs the XX/XY genetic sex determination system. We show here that the phenotypic sex of medaka is affected by changes in lipid metabolism. Medaka larvae subjected to 5 days of starvation underwent female-to-male sex reversal. Metabolomic and RT-qPCR analyses indicated that pantothenate metabolism was suppressed by starvation. Consistently, inhibiting the pantothenate metabolic pathway caused sex reversal. The final metabolite in this pathway is coenzyme A, an essential factor for lipogenesis. Inhibiting fatty acid synthesis, the first step of lipogenesis, also caused sex reversal. The expression of dmrt1, a critical gene for male development, was suppressed by starvation, and a dmrt1 (13) mutant did not show sex reversal under starvation. Collectively, these results indicate that fatty acid synthesis is involved in female-to-male sex reversal through ectopic expression of male gene dmrt1 under starvation.

PURPOSE

We aimed to evaluate the efficacy and safety of use of the Fasting Algorithm for Singaporeans with Type 2 Diabetes (FAST) during Ramadan.

PURPOSE

General practitioners (GPs) are part of the US physician workforce, but little is known about who they are, what they do, and how they differ from family physicians (FPs). We describe self-identified GPs and compare them with board-certified FPs.

PURPOSE

Although cesarean delivery is the most common surgical procedure in the United States, postoperative opioid prescribing varies greatly. We hypothesized that patient characteristics, procedural characteristics, or both would be associated with high vs low opioid use after discharge. This information could help individualize prescriptions.

Background and objectives

Tolvaptan is approved to slow kidney function decline in adults with autosomal dominant polycystic kidney disease (ADPKD) at risk of rapid progression. Because in vitro studies indicated that the tolvaptan oxobutyric acid metabolite inhibits organic anion–transporting polypeptide (OATP)1B1 and OATP1B3, United States prescribing information advises avoiding concurrent use with OATP1B1/1B3 substrates, including hepatic hydroxymethyl glutaryl–CoA reductase inhibitors (statins). This post hoc analysis of the pivotal phase 3 tolvaptan trials (Tolvaptan Efficacy and Safety in Management of Autosomal Dominant Polycystic Kidney Disease and Its Outcomes [TEMPO] 3:4 trial [NCT00428948] and Replicating Evidence of Preserved Renal Function: an Investigation of Tolvaptan Safety and Efficacy in ADPKD [REPRISE] trial [NCT02160145]) examined the safety of concurrent tolvaptan/statin use.

T-cell receptor (TCR)–based therapeutic cells and agents have emerged as a new class of effective cancer therapies. These therapies work on cells that express intracellular cancer-associated proteins by targeting peptides displayed on MHC receptors. However, cross-reactivities of these agents to off-target cells and tissues have resulted in serious, sometimes fatal, adverse events. We have developed a high-throughput genetic platform (termed "PresentER") that encodes MHC-I peptide minigenes for functional immunologic assays and determines the reactivities of TCR-like therapeutic agents against large libraries of MHC-I ligands. In this article, we demonstrated that PresentER could be used to identify the on-and-off targets of T cells and TCR-mimic (TCRm) antibodies using in vitro coculture assays or binding assays. We found dozens of MHC-I ligands that were cross-reactive with two TCRm antibodies and two native TCRs and that were not easily predictable by other methods.

Merkel cell carcinoma (MCC) is often caused by persistent expression of Merkel cell polyomavirus (MCPyV) T-antigen (T-Ag). These non-self proteins comprise about 400 amino acids (AA). Clinical responses to immune checkpoint inhibitors, seen in about half of patients, may relate to T-Ag–specific T cells. Strategies to increase CD8⁺ T-cell number, breadth, or function could augment checkpoint inhibition, but vaccines to augment immunity must avoid delivery of oncogenic T-antigen domains. We probed MCC tumor-infiltrating lymphocytes (TIL) with an artificial antigen-presenting cell (aAPC) system and confirmed T-Ag recognition with synthetic peptides, HLA-peptide tetramers, and dendritic cells (DC). TILs from 9 of 12 (75%) subjects contained CD8⁺ T cells recognizing 1–8 MCPyV epitopes per person. Analysis of 16 MCPyV CD8⁺ TIL epitopes and prior TIL data indicated that 97% of patients with MCPyV⁺ MCC had HLA alleles with the genetic potential that restrict CD8⁺ T-cell responses to MCPyV T-Ag. The LT AA 70–110 region was epitope rich, whereas the oncogenic domains of T-Ag were not commonly recognized. Specific recognition of T-Ag–expressing DCs was documented. Recovery of MCPyV oncoprotein–specific CD8⁺ TILs from most tumors indicated that antigen indifference was unlikely to be a major cause of checkpoint inhibition failure. The myriad of epitopes restricted by diverse HLA alleles indicates that vaccination can be a rational component of immunotherapy if tumor immune suppression can be overcome, and the oncogenic regions of T-Ag can be modified without impacting immunogenicity.

Vaccinia virus (VACV) is a double-stranded DNA virus that devotes a large portion of its 200 kbp genome to suppressing and manipulating the immune response of its host. Here, we investigated how targeted removal of immunomodulatory genes from the VACV genome impacted immune cells in the tumor microenvironment with the intention of improving the therapeutic efficacy of VACV in breast cancer. We performed a head-to-head comparison of six mutant oncolytic VACVs, each harboring deletions in genes that modulate different cellular pathways, such as nucleotide metabolism, apoptosis, inflammation, and chemokine and interferon signaling. We found that even minor changes to the VACV genome can impact the immune cell compartment in the tumor microenvironment. Viral genome modifications had the capacity to alter lymphocytic and myeloid cell compositions in tumors and spleens, PD-1 expression, and the percentages of virus-targeted and tumor-targeted CD8⁺ T cells. We observed that while some gene deletions improved responses in the nonimmunogenic 4T1 tumor model, very little therapeutic improvement was seen in the immunogenic HER2/neu TuBo model with the various genome modifications. We observed that the most promising candidate genes for deletion were those that interfere with interferon signaling. Collectively, this research helped focus attention on the pathways that modulate the immune response in the context of VACV oncolytic virotherapy. They also suggest that the greatest benefits to be obtained with these treatments may not always be seen in "hot tumors."

We conducted a 7-year case–control study of people ≥30 years of age on the prevalence of influenza, gastroenteritis, hepatitis, and pneumonia infections to indirectly examine whether these infections correlated to malignant cancer formation. Data were extracted from a large medical claims database of a Japanese social health insurance system; the case group included 2,354 people with their first cancer diagnosis in the 7th year of this study, and the control group included 48,395 people with no cancer diagnosis by the 7th year. The yearly prevalence rates of influenza, gastroenteritis, hepatitis, and pneumonia infections increased throughout the study period. Age-adjusted ORs and 95% confidence intervals (CI) in cases 1 year before cancer detection were significantly higher—for influenza 1.29 (95% CI, 1.14–1.46), for gastroenteritis 1.60 (95% CI, 1.41–1.82), for hepatitis 3.38 (95% CI, 2.12–5.37), for pneumonia 2.36 (95% CI, 1.79–3.13), and for any of these four diseases 1.55 (95% CI, 1.40–1.70). In influenza infections, significant ORs were found only in the 2nd and 6th years before cancer diagnosis. For each cancer site, an increased rate of infection prior to cancer diagnosis was observed. Here, we showed that increased infections during the precancerous stage, a possible surrogate for tumor-induced immune suppression, correlated to eventual cancer development.

Fnr is a transcriptional regulator that controls the expression of a variety of genes in response to oxygen limitation in bacteria. Genome sequencing revealed four genes (fnr1, fnr3, fnr5, and fnr7) coding for Fnr proteins in Paenibacillus polymyxa WLY78. Fnr1 and Fnr3 showed more similarity to each other than to Fnr5 and Fnr7. Also, Fnr1 and Fnr3 exhibited high similarity with Bacillus cereus Fnr and Bacillus subtilis Fnr in sequence and structures. Both the aerobically purified His-tagged Fnr1 and His-tagged Fnr3 in Escherichia coli could bind to the specific DNA promoter. Deletion analysis showed that the four fnr genes, especially fnr1 and fnr3, have significant impacts on growth and nitrogenase activity. Single deletion of fnr1 or fnr3 led to a 50% reduction in nitrogenase activity, and double deletion of fnr1 and fnr3 resulted to a 90% reduction in activity. Genome-wide transcription analysis showed that Fnr1 and Fnr3 indirectly activated expression of nif (nitrogen fixation) genes and Fe transport genes under anaerobic conditions. Fnr1 and Fnr3 inhibited expression of the genes involved in the aerobic respiratory chain and activated expression of genes responsible for anaerobic electron acceptor genes.

IMPORTANCE The members of the nitrogen-fixing Paenibacillus spp. have great potential to be used as a bacterial fertilizer in agriculture. However, the functions of the fnr gene(s) in nitrogen fixation and other metabolisms in Paenibacillus spp. are not known. Here, we found that in P. polymyxa WLY78, Fnr1 and Fnr3 were responsible for regulation of numerous genes in response to changes in oxygen levels, but Fnr5 and Fnr7 exhibited little effect. Fnr1 and Fnr3 indirectly or directly regulated many types of important metabolism, such as nitrogen fixation, Fe uptake, respiration, and electron transport. This study not only reveals the function of the fnr genes of P. polymyxa WLY78 in nitrogen fixation and other metabolisms but also will provide insight into the evolution and regulatory mechanisms of fnr in Paenibacillus.