Consistencies in the dietary and isotopic niche of spotted seatrout, Cynoscion nebulosus, across a salinity gradient within a coastal Louisiana estuary.

Estuaries are essential habitats for recreational and commercial fish that are shaped by both natural and anthropogenic processes. In Louisiana a combination of climate change and planned coastal restoration actions is predicted to increase freshwater introduction to coastal estuaries. As such there is a need to quantify the relationships between estuarine fish ecology and salinity to aid in predicting how species will respond to shifts in salinity. We investigated the relative abundance and dietary niches of adult (24.5 ± 5.4 cm standard length) spotted seatrout Cynoscion nebulosus across varying salinity regimes (oligohaline, mesohaline, and polyhaline) within Barataria Bay, Louisiana, using a combination of net sampling and gut content and stable isotopes analysis. We found that the relative abundance of C. nebulosus was lowest at the oligohaline site, translating to approximately five fewer fish captured for every single psu decrease in a site's average annual salinity. In contrast, we found that diets and, to a lesser extent, isotopic niches had a high degree of overlap across sites with differing salinity regimes. Fish and penaeid shrimp were the most common and important prey taxa recovered from guts at all sites. The small isotopic differences found among sites were likely due to spatial variation in hydrogeochemical baselines, and the observed isotopic overlap provides support for the idea that C. nebulosus move between adjacent salinity regimes and forage throughout Barataria Bay. Our results contribute to a greater understanding of the salinity preference and trophic ecology of C. nebulosus that can aid in predicting their responses to future salinity and habitat changes within Barataria Bay associated with predicted climate change and planned coastal restoration actions.

Culturing and Imaging Glioma Stem Cells in 3D Collagen Matrices.

Methods to maintain human glioma stem cells as neurosphere cultures and image their dynamic behavior in 3D collagen matrices are described. Additional approaches to monitor glioma stem cell differentiation into mesenchymal-type cells, along with example data are included. Together, these approaches enable glioma stem cell differentiation to be controlled while maintaining the cells in culture, as well as allowing cell dynamics to be captured and analyzed. These methods should be helpful for those seeking to understand the molecular mechanisms driving the invasion of glioma cells through three-dimensional environments. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Culturing human glioma stem cells as neurospheres Basic Protocol 2: Inducing GSC adherence and monitoring their differentiation into mesenchymal cells Support Protocol 1: Preparing fibronectin-coated dishes for cell microscopy Basic Protocol 3: Embedding GSCs in a 3D collagen matrix to study their invasive behavior Support Protocol 2: Phase-contrast imaging with a tiled matrix to study cell migration in a 3D gel.

Non-muscle myosin II and the plasticity of 3D cell migration.

Confined cells migrating through 3D environments are also constrained by the laws of physics, meaning for every action there must be an equal and opposite reaction for cells to achieve motion. Fascinatingly, there are several distinct molecular mechanisms that cells can use to move, and this is reflected in the diverse ways non-muscle myosin II (NMII) can generate the mechanical forces necessary to sustain 3D cell migration. This review summarizes the unique modes of 3D migration, as well as how NMII activity is regulated and localized within each of these different modes. In addition, we highlight tropomyosins and septins as two protein families that likely have more secrets to reveal about how NMII activity is governed during 3D cell migration. Together, this information suggests that investigating the mechanisms controlling NMII activity will be helpful in understanding how a single cell transitions between distinct modes of 3D migration in response to the physical environment.

Phenotypic continuum of NFU1-related disorders.

Bi-allelic variants in Iron-Sulfur Cluster Scaffold (NFU1) have previously been associated with multiple mitochondrial dysfunctions syndrome 1 (MMDS1) characterized by early-onset rapidly fatal leukoencephalopathy. We report 19 affected individuals from 10 independent families with ultra-rare bi-allelic NFU1 missense variants associated with a spectrum of early-onset pure to complex hereditary spastic paraplegia (HSP) phenotype with a longer survival (16/19) on one end and neurodevelopmental delay with severe hypotonia (3/19) on the other. Reversible or irreversible neurological decompensation after a febrile illness was common in the cohort, and there were invariable white matter abnormalities on neuroimaging. The study suggests that MMDS1 and HSP could be the two ends of the NFU1-related phenotypic continuum.

Prospective Assessment of Clinical Tests Used to Evaluate Tibial Stress Fracture.

Background: Tibial stress fracture (SFx) is the most common SFx of the lower extremity. Presently, diagnostic accuracy of clinical examination techniques for tibial SFx remains suboptimal. Purpose: To assess the diagnostic effectiveness of 5 clinical tests for tibial SFx individually versus a test item cluster. Study Design: Cohort study (diagnosis); Level of evidence, 3. Methods: A total of 50 patients with tibial pain (17 with bilateral symptoms) were assessed with 5 clinical examination tests (tibial fulcrum test, focal tenderness to palpation, heel percussion test, therapeutic ultrasound test, and 128-Hz tuning fork test) before they underwent diagnostic imaging (radionuclide bone scan). The application of the clinical tests was counterbalanced to minimize the likelihood of carryover effects. Patients provided a pain rating immediately before and after the application of each clinical test. Results: The prevalence of tibial SFx among the study participants was 52.2%. High levels of specificity were produced by the therapeutic ultrasound test (93.8%), tuning fork test (90.6%), and percussion test (90.6%). The fulcrum test had moderate to high specificity (84.4%). All tests demonstrated low levels of sensitivity, with the highest levels found for focal tenderness to palpation (48.6%) and fulcrum (45.7%). The fulcrum test provided the highest positive likelihood ratio (2.93), followed by the therapeutic ultrasound test (2.30). The fulcrum test had the lowest negative likelihood ratio (0.64), with the focal tenderness to palpation and tuning fork tests having negative likelihood ratios >1.0. Combinations of these clinical tests did not improve the prediction of tibial SFx above that observed among the individual tests. Conclusion: The clinical tests evaluated were generally highly specific, but all had low sensitivity. The fulcrum test provided the highest level of diagnostic accuracy; however, it was inadequate for definitive clinical management. Combining tests did not improve the diagnostic accuracy of tibial SFx.

Tackling antimicrobial stewardship through synergy and antimicrobial peptides.

The unrestricted use of antibiotics has led to rapid development of antibiotic resistance (AR) and renewed calls to address this serious problem. This review summarizes the most common mechanisms of antibiotic action, and in turn antibiotic resistance, as well as pathways to mitigate the harm. Focus is then turned to emerging antibiotic strategies, including antimicrobial peptides (AMPs), with a discussion of their modes of action, biochemical features, and potential challenges for their use as antibiotics. The role of synergy in antimicrobials is also examined, with a focus on the synergy of AMPs and other emerging interactions with synergistic potential.

Histidine Ligated Iron-Sulfur Peptides.

Iron-sulfur clusters are thought to be ancient cofactors that could have played a role in early protometabolic systems. Thus far, redox active, prebiotically plausible iron-sulfur clusters have always contained cysteine ligands to the cluster. However, extant iron-sulfur proteins can be found to exploit other modes of binding, including ligation by histidine residues, as seen with [2Fe-2S] Rieske and MitoNEET proteins. Here, we investigated the ability of cysteine- and histidine-containing peptides to coordinate a mononuclear Fe2+ center and a [2Fe-2S] cluster and compare their properties with purified iron-sulfur proteins. The iron-sulfur peptides were characterized by UV-vis, circular dichroism, and paramagnetic NMR spectroscopies and cyclic voltammetry. Small (≤6 amino acids) peptides can coordinate [2Fe-2S] clusters through a combination of cysteine and histidine residues with similar reduction potentials as their corresponding proteins. Such complexes may have been important for early cell-like systems.

COVID-19-related healthcare impacts: an uncontrolled, segmented time-series analysis of tuberculosis diagnosis services in Mozambique, 2017-2020.

INTRODUCTION: Currently, COVID-19 dominates the public health agenda and poses a permanent threat, leading to health systems' exhaustion and unprecedented service disruption. Primary healthcare services, including tuberculosis services, are at increased risk of facing severe disruptions, particularly in low-income and middle-income countries. Indeed, corroborating model-based forecasts, there is increasing evidence of the COVID-19 pandemic's negative impact on tuberculosis case detection. METHODS: Applying a segmented time-series analysis, we assessed the effects of COVID-19-related measures on tuberculosis diagnosis service across districts in Mozambique. Ministry health information system data were used from the first quarter of 2017 to the end of 2020. The model, performed under the Bayesian premises, was estimated as a negative binomial with random effects for districts and provinces. RESULTS: A total of 154 districts were followed for 16 consecutive quarters. Together, these districts reported 96 182 cases of all forms of tuberculosis in 2020. At baseline (first quarter of 2017), Mozambique had an estimated incidence rate of 283 (95% CI 200 to 406) tuberculosis cases per 100 000 people and this increased at a 5% annual rate through the end of 2019. We estimated that 17 147 new tuberculosis cases were potentially missed 9 months after COVID-19 onset, resulting in a 15.1% (95% CI 5.9 to 24.0) relative loss in 2020. The greatest impact was observed in the southern region at 40.0% (95% CI 30.1 to 49.0) and among men at 15% (95% CI 4.0 to 25.0). The incidence of pulmonary tuberculosis increased at an average rate of 6.6% annually; however, an abrupt drop (15%) was also observed immediately after COVID-19 onset in March 2020. CONCLUSION: The most significant impact of the state of emergency was observed between April and June 2020, the quarter after COVID-19 onset. Encouragingly, by the end of 2020, clear signs of health system recovery were visible despite the initial shock.

Cytoplasmic pressure maintains epithelial integrity and inhibits cell motility.

Cytoplasmic pressure, a function of actomyosin contractility and water flow, can regulate cellular morphology and dynamics. In mesenchymal cells, cytoplasmic pressure powers cell protrusion through physiological three-dimensional extracellular matrices. However, the role of intracellular pressure in epithelial cells is relatively unclear. Here we find that high cytoplasmic pressure is necessary to maintain barrier function, one of the hallmarks of epithelial homeostasis. Further, our data show that decreased cytoplasmic pressure facilitates lamellipodia formation during the epithelial to mesenchymal transition (EMT). Critically, activation of the actin nucleating protein Arp2/3 is required for the reduction in cytoplasmic pressure and lamellipodia formation in response to treatment with hepatocyte growth factor (HGF) to induce EMT. Thus, elevated cytoplasmic pressure functions to maintain epithelial tissue integrity, while reduced cytoplasmic pressure triggers lamellipodia formation and motility during HGF-dependent EMT.

G-quadruplex targeting chemical nucleases as a nonperturbative tool for analysis of cellular G-quadruplex DNA.

G-quadruplex structures are associated with various biological activities, while in vivo evidence is essential to confirm the formation of G-quadruplexes inside cells. Most conventional agents that recognize G-quadruplex, including antibodies and small-molecule G-quadruplex ligands, either stabilize the G-quadruplex or prevent G-quadruplex unfolding by helicase, thereby artificially increasing the G-quadruplex levels in cells. Unambiguous study of G-quadruplexes at natural cellular levels requires agents that do not enhance the stability of G-quadruplex. Herein, we report the first example of nonperturbative chemical nucleases that do not influence the stability of G-quadruplex telomeric DNA but can selectively cleave G-quadruplex DNA over duplex DNA. These chemical nucleases can be readily taken up by cells and promote selective cleavage of telomeric DNA with low levels of nonselective DNA cleavage of other regions of the genome. The cleavage of G-quadruplex telomeric DNA by nonperturbative chemical nucleases confirms the formation of G-quadruplex telomeric DNA in live cells.

Enhanced Synergism and Mechanism of Action Studies of Synthetic Antimicrobial Metallopeptides.

Antimicrobial peptides (AMPs) are found throughout most kingdoms of life, are an important part of host immunity, and have been shown to act synergistically in various organisms to ameliorate bacterial infections. Herein, we report the synergistic behavior observed between two AMPs, Sub5 and CP10A, against E. coli. In addition, enhanced synergistic activity against E. coli and MRSA 43300 for two derivatives of Sub5, extended with the amino-terminal copper and nickel (ATCUN) binding motif, is observed when dosed together with CP10A, while displaying little cytotoxicity towards human dermal fibroblasts. All three combinations of peptides co-localized within bacterial cells as evidenced by fluorescence confocal microscopy. Investigations into the mechanism of synergy shows that all peptides indirectly damage DNA within cells, while only the ATCUN derivatives can oxidize phospholipids. Combinations of peptides were also shown to upregulate the concentration of reactive oxygen species within both E. coli and MRSA 43300. These results suggest that the production of reactive oxygen species is an important aspect mechanistically and further highlights the potential of these metallopeptides to aid in the treatment of antibiotic-resistant infections.

Cluster exchange reactivity of [2Fe-2S]-bridged heterodimeric BOLA1-GLRX5.

Mitochondrial BOLA1 is known to form a [2Fe-2S] cluster-bridged heterodimeric complex with mitochondrial monothiol glutaredoxin GLRX5; however, the function of this heterodimeric complex is unclear. Some reports suggest redundant roles for BOLA1 and a related protein, BOLA3, with both involved in the maturation of [4Fe-4S] clusters in a subset of mitochondrial proteins. However, a later report on the structure of BOLA1-GLRX5 heterodimeric complex demonstrated a buried cluster environment and predicted a redox role instead of the cluster trafficking role suggested for the BOLA3-GLRX5 heterodimeric complex. Herein, we describe a detailed kinetic study of relative cluster exchange reactivity involving heterodimeric complex of BOLA1 with GLRX5. By the use of CD spectroscopy, it is demonstrated that [2Fe-2S]-bridged BOLA1-GLRX5 can be readily formed by cluster uptake from donors such as ISCU or [2Fe-2S](GS)4 complex, but not from ISCA1 or ISCA2. Rapid holo-formation following delivery from [2Fe-2S](GS)4 supports possible physiological relevance in the cellular labile iron pool. Holo [2Fe-2S] BOLA1-GLRX5 heterodimeric complex is incapable of donating cluster to apo protein acceptors, providing experimental support for a nontrafficking role. Finally, we report the formation and reactivity of the holo [2Fe-2S]-bridged BOLA1 homodimer (lacking a partner GLRX). While the holo-heterodimer is thermodynamically more stable, by contrast the holo BOLA1 homodimer does demonstrate facile cluster exchange reactivity.

Public Health Response to Zika Virus Exposure of Air Force Members Deployed to Caribbean Islands, 2016.

INTRODUCTION: The emergence of Zika virus disease (ZVD) in areas of military operations provided a new opportunity for force health protection. ZVD infection had an estimated 4:1 asymptomatic-to-symptomatic ratio and can cause neurologic sequelae. MATERIALS AND METHODS: We provide a brief report of a field investigation utilizing laboratory-based surveillance and survey instruments to characterize ZVD risk among personnel deployed to the Dominican Republic in support of Operation NEW HORIZONS (NH). Additionally, we describe a cluster of 3 ZVD cases among 8 aircrew on a short mission to St. Croix (U.S. Virgin Islands). RESULTS: Following Operation NH, 6 of a total 189 deployed cohort members tested positive for ZVD by immunoglobulin M and confirmatory plaque reduction neutralization test (3.2%). Reverse transcription polymerase chain reaction testing in urine or serum was positive in 4 of those 6 cases. All 6 cases reported at least one symptom, with 5 reporting subjective fever and arthralgia and 4 reporting rash. Cases were less likely to have air-conditioned living quarters (odds ratio = 0.1; 95% confidence interval 0.02-0.77; P < 0.03), but were otherwise similar to non-cases. Likewise, in St. Croix, 3/8 tested positive by immunoglobulin M and plaque reduction neutralization test for an attack rate of 38%. Similar to Operation NH, all three cases were symptomatic with subjective fever (67%), arthralgia (67%), and/or rash (100%). CONCLUSIONS: This field investigation identified differing, mission location-dependent ZVD attack rates and a 0:9 asymptomatic-to-symptomatic case ratio. As this was unexpected based on a previous report of a 4:1 ratio, it emphasizes the need to be cautious before generalizing outbreak characteristics between populations while also offering additional practical experience for force health protection.

Correction: Copper(ii) l/d-valine-(1,10-phen) complexes target human telomeric G-quadruplex motifs and promote site-specific DNA cleavage and cellular cytotoxicity.

Correction for 'Copper(ii) l/d-valine-(1,10-phen) complexes target human telomeric G-quadruplex motifs and promote site-specific DNA cleavage and cellular cytotoxicity' by Farukh Arjmand et al., Dalton Trans., 2020, 49, 9888-9899, DOI: 10.1039/d0dt01527j.

Copper(ii) l/d-valine-(1,10-phen) complexes target human telomeric G-quadruplex motifs and promote site-specific DNA cleavage and cellular cytotoxicity.

Chiral l-/d-valine-(1,10-phen)-Cu(ii) complexes that target G-quadruplex DNA were synthesized and thoroughly characterized by UV-vis, IR, EPR, ESI-MS, elemental analysis and single crystal X-ray spectroscopy. Complexes 1a and 1b crystallized in the monoclinic P21/c and C2 space groups, respectively. On the basis of Wolfe-Shimer analyses, the binding affinities of 1a and 1b with G-quadruplex telomeric DNA were determined, and 1a exhibited significantly higher binding as compared to 1b. Site selective cleavage of G4-DNA was demonstrated by employing the time-dependent PAGE assay, with 1a exhibiting a significantly higher cleavage rate from A1 to G22 (4.32 (±0.13) µM h-1) than 1b (4.29 (±0.11) µM h-1). The DNA cleavage profile demonstrated that both complexes perform non-random double-strand cleavage by following first-order kinetics (kobs = 0.9432 min-1 for 1a and kobs = 0.6574 min-1 for 1b). Molecular docking simulations were performed with both parallel and anti-parallel topologies of the quadruplex to provide a clear insight on G-quadruplex-complex interactions. Complexes 1a and 1b were found to interact strongly at the minor groove cavity of the quadruplex with preferential selectivity for the parallel vs. anti-parallel quadruplex. The cytotoxic activities of complexes 1a and 1b were evaluated on a few notably important human cancer cell lines, viz, breast (MCF-7), pancreatic strains (BxPC3, AsPC1) and liver (Huh7) by an MTT assay. Both 1a and 1b exhibited pronounced cytotoxic activity with remarkably low IC50 values (1-3 µM) for all tested cancer strains.

Artificial Metalloenzymes: Recent Developments and Innovations in Bioinorganic Catalysis.

Cellular life is orchestrated by the biochemical components of cells that include nucleic acids, lipids, carbohydrates, proteins, and cofactors such as metabolites and metals, all of which coalesce and function synchronously within the cell. Metalloenzymes allow for such complex chemical processes, as they catalyze a myriad of biochemical reactions both efficiently and selectively, where the metal cofactor provides additional functionality to promote reactivity not readily achieved in their absence. While the past 60 years have yielded considerable insight on how enzymes catalyze these reactions, a need to engineer and develop artificial metalloenzymes has been driven not only by industrial and therapeutic needs, but also by innate human curiosity. The design of miniature enzymes, both rationally and through serendipity, using both organic and inorganic building blocks has been explored by many scientists over the years and significant progress has been made. Herein, recent developments over the past 5 years in areas that have not been recently reviewed are summarized, and prospects for future research in these areas are addressed.

Characterization of [2Fe-2S]-Cluster-Bridged Protein Complexes and Reaction Intermediates by use of Native Mass Spectrometric Methods.

Many iron-sulfur proteins involved in cluster trafficking form [2Fe-2S]-cluster-bridged complexes that are often challenging to characterize because of the inherent instability of the cluster at the interface. Herein, we illustrate the use of fast, online buffer exchange coupled to a native mass spectrometry (OBE nMS) method to characterize [2Fe-2S]-cluster-bridged proteins and their transient cluster-transfer intermediates. The use of this mechanistic and protein-characterization tool is demonstrated with holo glutaredoxin 5 (GLRX5) homodimer and holo GLRX5:BolA-like protein 3 (BOLA3) heterodimer. Using the OBE nMS method, cluster-transfer reactions between the holo-dimers and apo-ferredoxin (FDX2) are monitored, and intermediate [2Fe-2S] species, such as (FDX2:GLRX5:[2Fe-2S]:GSH) and (FDX2:BOLA3:GLRX5:[2Fe-2S]:GSH) are detected. The OBE nMS method is a robust technique for characterizing iron-sulfur-cluster-bridged protein complexes and transient iron-sulfur-cluster transfer intermediates.

The MyMOMA domain of MYO19 encodes for distinct Miro-dependent and Miro-independent mechanisms of interaction with mitochondrial membranes.

MYO19 interacts with mitochondria through a C-terminal membrane association domain (MyMOMA). Specific mechanisms for localization of MYO19 to mitochondria are poorly understood. Using promiscuous biotinylation data in combination with existing affinity-capture databases, we have identified a number of putative MYO19-interacting proteins. We chose to explore the interaction between MYO19 and the mitochondrial GTPase Miro2 by expressing mchr-Miro2 in combination with GFP-tagged fragments of the MyMOMA domain and assaying for recruitment of MYO19-GFP to mitochondria. Coexpression of MYO19898-970 -GFP with mchr-Miro2 enhanced MYO19898-970 -GFP localization to mitochondria. Mislocalizing Miro2 to filopodial tips or the cytosolic face of the nuclear envelope did not recruit MYO19898-970 -GFP to either location. To address the kinetics of the Miro2/MYO19 interaction, we used FRAP analysis and permeabilization-activated reduction in fluorescence analysis. MyMOMA constructs containing a putative membrane-insertion motif but lacking the Miro2-interacting region displayed slow exchange kinetics. MYO19898-970 -GFP, which does not include the membrane-insertion motif, displayed rapid exchange kinetics, suggesting that MYO19 interacting with Miro2 has higher mobility than MYO19 inserted into the mitochondrial outer membrane. Mutation of well-conserved, charged residues within MYO19 or within the switch I and II regions of Miro2 abolished the enhancement of MYO19898-970 -GFP localization in cells ectopically expressing mchr-Miro2. Additionally, expressing mutant versions of Miro2 thought to represent particular nucleotide states indicated that the enhancement of MYO19898-970 -GFP localization is dependent on Miro2 nucleotide state. Taken together, these data suggest that membrane-inserted MYO19 is part of a larger complex, and that Miro2 plays a role in integration of actin- and microtubule-based mitochondrial activities.