The Effects of Protein Kinase C Beta II Peptide Modulation on Superoxide Release in Rat Polymorphonuclear Leukocytes

Phorbol 12-myristate 13-acetate (PMA; a diacylglycerol mimetic) is known to augment polymorphonuclear leukocyte (PMN) superoxide (SO) release via protein kinase C (PKC) activation. However, the role of PKC beta II (βII) mediating this response is not known. It’s known that myristic acid (myr-) conjugation facilitates intracellular delivery of the cargo sequence, and that putative PKCβII activator and inhibitor peptides work by augmenting or attenuating PKCβII translocation to cell membrane substrates (e.g. NOX-2). Therefore, we hypothesize that myr- conjugated PKCβII peptide-activator (N-myr-SVEIWD; myr-PKCβ+) would increase PMA-induced rat PMN SO release, whereas, myr-PKCβII peptide-inhibitor (N-myr-SLNPEWNET; myr-PKCβ-) would attenuate this response compared to non-drug treated controls. Rat PMNs (5x106) were incubated for 15min at 370C in the presence/absence of myr-PKCβ+/- (20 μM) or SO dismutase (SOD;10μg/mL; n=8) as positive control. PMA (100nM) induced PMN SO release was measured spectrophotometrically at 550nm via reduction of ferricytochrome c for 390 sec. PMN SO release increased absorbance to 0.39±0.04 in non-drug treated controls (n=28), and 0.49±0.05 in myr-PKCβ+(n=16). This response was significantly increased from 180 seconds to 240 seconds (p\u3c0.05). By contrast, myr-PKCβ- (0.26±0.03; n=14) significantly attenuated PMA-induced SO release compared to non-drug controls and myr-PKCβ+ (p\u3c0.05). SOD-treated samples showed \u3e90% reduction of PMA-induced SO release and was significantly different from all groups (p\u3c0.01). Cell viability ranged between 94± to 98±2% in all groups as determined by 0.2% trypan blue exclusion. Preliminary results suggest that myr-PKCβ- significantly attenuates PMA-induced SO release, whereas myr-PKCβ+ significantly augments PMA-induced SO release, albeit transiently. Additional dose response and western blot experiments are planned with myr-PKCβ+/- in PMA-induced PMN SO release assays. This research was supported by the Department of Bio-Medical Sciences and the Division of Research at PCOM and by Young Therapeutics, LLC

Protein Kinase C Beta II Peptide Inhibitor Elicits Robust Effects on Attenuating Myocardial Ischemia/Reperfusion Injury

Reperfusion injury contributes to myocardial tissue damage following a heart attack partly due to the generation of reactive oxygen species (ROS) upon cardio-angioplasty. Protein kinase C beta II (PKCβII) inhibition during reperfusion with peptide inhibitor (N-myr-SLNPEWNET; PKCβII-) decreases ROS release and leukocyte infiltration in rat hind-limb and myocardial ischemia/reperfusion (I/R) studies, respectively. However, the role of activating PKCβII during reperfusion has not been previously determined. In this study, we hypothesize that myristoylated (myr)-PKCβII- will decrease infarct size and improve post-reperfused cardiac function compared to untreated controls, whereas PKCβII peptide activator (N-myr-SVEIWD; myr-PKCβII+) will show no improvement compared to control. Myristoylation of PKCβII peptides facilitate their entry into the cell in order to affect PKCβII activity by either augmenting or attenuating its translocation to cell membrane proteins, such as NOX-2. Isolated perfused rat hearts were subjected to global I(30min)/R(50min) and infused with myr-PKCβII+ (20μM; n=9), myr-PKCβII- (20µM; n=8), or plasma (control; n=9) at reperfusion. Hearts were frozen (-20oC), sectioned and stained using 1% triphenyltetrazolium chloride to differentiate necrotic tissue. The measurement of Left ventricular (LV) cardiac function was determined using a pressure transducer and infarct size was calculated as percent dead tissue vs. total heart tissue weight. Myr-PKCβII- significantly improved LV end-diastolic pressure 37±7 mmHg compared to control (58±5; p\u3c0.01) and myr-PKCβII+ (58±4; p\u3c0.01). Myr-PKCβII- significantly reduced infarct size to 14±3% compared to control (26±5%; p\u3c0.01), while myr-PKCβII+ (25±3%) showed no difference. The data indicate that myr-PKCβII- may be a putative treatment to reduce myocardial reperfusion injury when given to heart attack patients during cardio-angioplasty. Future studies are planned to determine infarct size by Image J analysis

Using lake sediments to assess the long-term impacts of anthropogenic activity in tropical river deltas

Tropical river deltas, and the social-ecological systems they sustain, are changing rapidly due to anthropogenic activity and climatic change. Baseline data to inform sustainable management options for resilient deltas is urgently needed and palaeolimnology (reconstructing past conditions from lake or wetland deposits) can provide crucial long-term perspectives needed to identify drivers and rates of change. We review how palaeolimnology can be a valuable tool for resource managers using three current issues facing tropical delta regions: hydrology and sediment supply, salinisation and nutrient pollution. The unique ability of palaeolimnological methods to untangle multiple stressors is also discussed. We demonstrate how palaeolimnology has been used to understand each of these issues, in other aquatic environments, to be incorporated into policy. Palaeolimnology is a key tool to understanding how anthropogenic influences interact with other environmental stressors, providing policymakers and resource managers with a ‘big picture’ view and possible holistic solutions that can be implemented

An internationally standardized species identification test for use on suspected seized rhinoceros horn in the illegal wildlife trade

Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).Rhinoceros (rhino) numbers have dwindled substantially over the past century. As a result, three of the five species are now considered to be critically endangered, one species is vulnerable and one species is near-threatened. Poaching has increased dramatically over the past decade due to a growing demand for rhino horn products, primarily in Asia. Improved wildlife forensic techniques, such as validated tests for species identification of seized horns, are critical to aid current enforcement and prosecution efforts and provide a deterrent to future rhino horn trafficking. Here, we present an internationally standardized species identification test based on a 230 base pair cytochrome-b region. This test improves on previous nested PCR protocols and can be used for the discrimination of samples with <20 pg of template DNA, thus suitable for DNA extracted from horn products. The assay was designed to amplify water buffalo samples, a common ‘rhino horn’ substitute, but to exclude human DNA, a common contaminant. Phylogenetic analyses using this partial cytochrome-b region resolved the five extant rhino species. Testing successfully returned a sequence and correct identification for all of the known rhino horn samples and vouchered rhino samples from museum and zoo collections, and provided species level identification for 47 out of 52 unknown samples from seizures. Validation and standardization was carried out across five different laboratories, in four different countries, demonstrating it to be an effective and reproducible test, robust to inter laboratory variation in equipment and consumables (such as PCR reagents). This is one of the first species identification tests to be internationally standardized to produce data for evidential proceedings and the first published validated test for rhinos, one of the flagship species groups of the illegal wildlife trade and for which forensic tools are urgently required. This study serves as a model for how species identification tests should be standardized and disseminated for wildlife forensic testing

Complete genomic sequences of Propionibacterium freudenreichii phages from Swiss cheese reveal greater diversity than Cutibacterium (formerly Propionibacterium) acnes phages

Background:A remarkable exception to the large genetic diversity often observed for bacteriophages infecting aspecific bacterial host was found for the Cutibacterium acnes (formerly Propionibacterium acnes) phages, which are highly homogeneous. Phages infecting the related species, which is also a member of the Propionibacteriaceae family, Propionibacterium freudenreichii , a bacterium used in production of Swiss-type cheeses, have also been described and are common contaminants of the cheese manufacturing process. However, little is known about their genetic composition and diversity.Results:We obtained seven independently isolated bacteriophages that infect P. freudenreichii from Swiss-typecheese samples, and determined their complete genome sequences. These data revealed that all seven phage isolates are of similar genomic length and GC% content, but their genomes are highly diverse, including genes encoding the capsid, tape measure, and tail proteins. In contrast to C. acnes phages, all P. freudenreichii phage genomes encode a putative integrase protein, suggesting they are capable of lysogenic growth. This is supported by the finding of related prophages in some P. freudenreichii strains. The seven phages could further be distinguished as belonging to two distinct genomic types, or ‘ clusters ’ , based on nucleotide sequences, and host range analyses conducted on a collection of P. freudenreichii strains show a higher degree of host specificity than is observed for the C. acnes phages.Conclusions:Overall, our data demonstrate P. freudenreichii bacteriophages are distinct from C. acnes phages, asevidenced by their higher genetic diversity, potential for lysogenic growth, and more restricted host ranges. This suggests substantial differences in the evolution of these related species from the Propionibacteriaceae family and their phages, which is potentially related to their distinct environmental niches

Treg and CTLA-4: Two intertwining pathways to immune tolerance.

Both the CTLA-4 pathway and regulatory T cells (Treg) are essential for the control of immune homeostasis. Their therapeutic relevance is highlighted by the increasing use of anti-CTLA-4 antibody in tumor therapy and the development of Treg cell transfer strategies for use in autoimmunity and transplantation settings. The CTLA-4 pathway first came to the attention of the immunological community in 1995 with the discovery that mice deficient in Ctla-4 suffered a fatal lymphoproliferative syndrome. Eight years later, mice lacking the critical Treg transcription factor Foxp3 were shown to exhibit a remarkably similar phenotype. Much of the debate since has centered on the question of whether Treg suppressive function requires CTLA-4. The finding that it does in some settings but not in others has provoked controversy and inevitable polarization of opinion. In this article, I suggest that CTLA-4 and Treg represent complementary and largely overlapping mechanisms of immune tolerance. I argue that Treg commonly use CTLA-4 to effect suppression, however CTLA-4 can also function in the non-Treg compartment while Treg can invoke CTLA-4-independent mechanisms of suppression. The notion that Foxp3 and CTLA-4 direct independent programs of immune regulation, which in practice overlap to a significant extent, will hopefully help move us towards a better appreciation of the underlying biology and therapeutic significance of these pathways

Overseas Treatment of Latent Tuberculosis Infection in US-Bound Immigrants.

Seventy percent of tuberculosis (TB) cases in the United States occur among non-US-born persons; cases usually result from reactivation of latent TB infection (LTBI) likely acquired before the person's US arrival. We conducted a prospective study among US immigrant visa applicants undergoing the required overseas medical examination in Vietnam. Consenting applicants >15 years of age were offered an interferon-γ release assay (IGRA); those 12-14 years of age received an IGRA as part of the required examination. Eligible participants were offered LTBI treatment with 12 doses of weekly isoniazid and rifapentine. Of 5,311 immigrant visa applicants recruited, 2,438 (46%) consented to participate; 2,276 had an IGRA processed, and 484 (21%) tested positive. Among 452 participants eligible for treatment, 304 (67%) initiated treatment, and 268 (88%) completed treatment. We demonstrated that using the overseas medical examination to provide voluntary LTBI testing and treatment should be considered to advance US TB elimination efforts

Cell-Cell Transmission Enables HIV-1 to Evade Inhibition by Potent CD4bs Directed Antibodies

HIV is known to spread efficiently both in a cell-free state and from cell to cell, however the relative importance of the cell-cell transmission mode in natural infection has not yet been resolved. Likewise to what extent cell-cell transmission is vulnerable to inhibition by neutralizing antibodies and entry inhibitors remains to be determined. Here we report on neutralizing antibody activity during cell-cell transmission using specifically tailored experimental strategies which enable unambiguous discrimination between the two transmission routes. We demonstrate that the activity of neutralizing monoclonal antibodies (mAbs) and entry inhibitors during cell-cell transmission varies depending on their mode of action. While gp41 directed agents remain active, CD4 binding site (CD4bs) directed inhibitors, including the potent neutralizing mAb VRC01, dramatically lose potency during cell-cell transmission. This implies that CD4bs mAbs act preferentially through blocking free virus transmission, while still allowing HIV to spread through cell-cell contacts. Thus providing a plausible explanation for how HIV maintains infectivity and rapidly escapes potent and broadly active CD4bs directed antibody responses in vivo

Global transpiration data from sap flow measurements : the SAPFLUXNET database

Plant transpiration links physiological responses of vegetation to water supply and demand with hydrological, energy, and carbon budgets at the land-atmosphere interface. However, despite being the main land evaporative flux at the global scale, transpiration and its response to environmental drivers are currently not well constrained by observations. Here we introduce the first global compilation of whole-plant transpiration data from sap flow measurements (SAPFLUXNET, https://sapfluxnet.creaf.cat/, last access: 8 June 2021). We harmonized and quality-controlled individual datasets supplied by contributors worldwide in a semi-automatic data workflow implemented in the R programming language. Datasets include sub-daily time series of sap flow and hydrometeorological drivers for one or more growing seasons, as well as metadata on the stand characteristics, plant attributes, and technical details of the measurements. SAPFLUXNET contains 202 globally distributed datasets with sap flow time series for 2714 plants, mostly trees, of 174 species. SAPFLUXNET has a broad bioclimatic coverage, with woodland/shrubland and temperate forest biomes especially well represented (80 % of the datasets). The measurements cover a wide variety of stand structural characteristics and plant sizes. The datasets encompass the period between 1995 and 2018, with 50 % of the datasets being at least 3 years long. Accompanying radiation and vapour pressure deficit data are available for most of the datasets, while on-site soil water content is available for 56 % of the datasets. Many datasets contain data for species that make up 90 % or more of the total stand basal area, allowing the estimation of stand transpiration in diverse ecological settings. SAPFLUXNET adds to existing plant trait datasets, ecosystem flux networks, and remote sensing products to help increase our understanding of plant water use, plant responses to drought, and ecohydrological processes. SAPFLUXNET version 0.1.5 is freely available from the Zenodo repository (https://doi.org/10.5281/zenodo.3971689; Poyatos et al., 2020a). The "sapfluxnetr" R package - designed to access, visualize, and process SAPFLUXNET data - is available from CRAN.Peer reviewe