Secondary Sites Research Articles

Carbohydrate-aromatic interface and molecular architecture of lignocellulose

Plant cell walls constitute the majority of lignocellulosic biomass and serve as a renewable resource of biomaterials and biofuel. Extensive interactions between polysaccharides and the aromatic polymer lignin make lignocellulose recalcitrant to enzymatic hydrolysis, but this polymer network remains poorly understood. Here we interrogate the nanoscale assembly of lignocellulosic components in plant stems using solid-state nuclear magnetic resonance and dynamic nuclear polarization approaches. We show that the extent of glycan-aromatic association increases sequentially across grasses, hardwoods, and softwoods. Lignin principally packs with the xylan in a non-flat conformation via non-covalent interactions and partially binds the junction of flat-ribbon xylan and cellulose surface as a secondary site. All molecules are homogeneously mixed in softwoods; this unique feature enables water retention even around the hydrophobic aromatics. These findings unveil the principles of polymer interactions underlying the heterogeneous architecture of lignocellulose, which may guide the rational design of more digestible plants and more efficient biomass-conversion pathways.

Discovery and Characterization of a Cryptic Secondary Binding Site in the Molecular Chaperone HSP70.

Heat Shock Protein 70s (HSP70s) are key molecular chaperones that are overexpressed in many cancers and often associated with metastasis and poor prognosis. It has proven difficult to develop ATP-competitive, drug-like small molecule inhibitors of HSP70s due to the flexible and hydrophilic nature of the HSP70 ATP-binding site and its high affinity for endogenous nucleotides. The aim of this study was to explore the potential for the inhibition of HSP70 through alternative binding sites using fragment-based approaches. A surface plasmon resonance (SPR) fragment screen designed to detect secondary binding sites in HSP70 led to the identification by X-ray crystallography of a cryptic binding site in the nucleotide-binding domain (NBD) of HSP70 adjacent to the ATP-binding site. Fragment binding was confirmed and characterized as ATP-competitive using SPR and ligand-observed NMR methods. Molecular dynamics simulations were applied to understand the interactions with the protein upon ligand binding, and local secondary structure changes consistent with interconversion between the observed crystal structures with and without the cryptic pocket were detected. A virtual high-throughput screen (vHTS) against the cryptic pocket was conducted, and five compounds with diverse chemical scaffolds were confirmed to bind to HSP70 with micromolar affinity by SPR. These results identified and characterized a new targetable site on HSP70. While targeting HSP70 remains challenging, the new site may provide opportunities to develop allosteric ATP-competitive inhibitors with differentiated physicochemical properties from current series.

A computational modeling of invadopodia protrusion into an extracellular matrix fiber network

Invadopodia are dynamic actin-rich membrane protrusions that have been implicated in cancer cell invasion and metastasis. In addition, invasiveness of cancer cells is strongly correlated with invadopodia formation, which are observed during extravasation and colonization of metastatic cancer cells at secondary sites. However, quantitative understanding of the interaction of invadopodia with extracellular matrix (ECM) is lacking, and how invadopodia protrusion speed is associated with the frequency of protrusion-retraction cycles remains unknown. Here, we present a computational framework for the characterization of invadopodia protrusions which allows two way interactions between intracellular branched actin network and ECM fibers network. We have applied this approach to predicting the invasiveness of cancer cells by computationally knocking out actin-crosslinking molecules, such as α-actinin, filamin and fascin. The resulting simulations reveal distinct invadopodia dynamics with cycles of protrusion and retraction. Specifically, we found that (1) increasing accumulation of MT1-MMP at tips of invadopodia as the duration of protrusive phase is increased, and (2) the movement of nucleus toward the leading edge of the cell becomes unstable as duration of the retractile phase (or myosin turnover time) is longer than 1 min.

Microfluidic deep mutational scanning of the human executioner caspases reveals differences in structure and regulation

The human caspase family comprises 12 cysteine proteases that are centrally involved in cell death and inflammation responses. The members of this family have conserved sequences and structures, highly similar enzymatic activities and substrate preferences, and overlapping physiological roles. In this paper, we present a deep mutational scan of the executioner caspases CASP3 and CASP7 to dissect differences in their structure, function, and regulation. Our approach leverages high-throughput microfluidic screening to analyze hundreds of thousands of caspase variants in tightly controlled in vitro reactions. The resulting data provides a large-scale and unbiased view of the impact of amino acid substitutions on the proteolytic activity of CASP3 and CASP7. We use this data to pinpoint key functional differences between CASP3 and CASP7, including a secondary internal cleavage site, CASP7 Q196 that is not present in CASP3. Our results will open avenues for inquiry in caspase function and regulation that could potentially inform the development of future caspase-specific therapeutics.

Primary non-Hodgkin's lymphoma of the thyroid, type MALT: a case report from the Adolphe SICE General Hospital, Pointe Noire (Congo - Brazzaville)

Primary lymphomas of the thyroid are rare tumours, representing less than 5% to 15% of thyroid cancers. These lymphomas develop on a pre-existing thyroid disease, notably Hashimoto's thyroiditis, or as a secondary site of a disseminated lymphoma. This pathology is aggressive and presents as a painful, rapidly evolving goitre with signs of compression. The most common histological types are non-Hodgkin's lymphoma type B. We report a case of thyroid non-Hodgkin's lymphoma of the MALT type in a 71 year old woman with a specific history; the positive diagnosis was made thanks to a histological study of the surgical specimen completed by an immunohistochemical study on paraffin sections. The postoperative clinical course after six years was favourable, with no local recurrence or distant metastasis. Keywords: Goiter; Primary Lymphoma; Thyroid; MALT; Immunohistochemistry

Water adsorption characteristic and its impact on pore structure and methane adsorption of various rank coals.

Coalbed methane not only is a new clean energy source, but also has potential damage to ecological environment. Water and methane coexist in coal reservoir; understanding the adsorption of water on coal and its impact on pore structure and methane adsorption of coal is vital to evaluate the reserves and productivity of coalbed methane. In the paper, water adsorption characteristics of various rank coals are firstly investigated by ten mathematical models. The modified Dent model provides a best fit, followed by GAB and Dent models. For GAB model, the primary site adsorption is more difficult to reach saturation, and the contribution rate of the secondary site adsorption is surprisingly high at P/P0 approaching 0, which can be attributed to the possible overestimation of GAB monolayer adsorption capacity and secondary site adsorption. Besides, the low-rank coal sample YZG2 exhibits more prominent hysteresis than middle- to high-rank coals. The low-pressure hysteresis can be attributed to the water-water interactions over the primary site and the strengthened binding forces of water molecules in the water desorption process. In contrast, the high-pressure hysteresis largely depends on pore structure of coal such as ink-bottle pores, especially for the studied sample YZG2. Besides, pore analyses by low-temperature nitrogen adsorption method show that the pre-adsorbed water has remarkable influence on micropores smaller than 10 nm, and the micropores smaller than 4 nm almost disappear for water-equilibrated coals, which is closely related to the formed water clusters and capillary water in pore throats. This finding reveals that more methane gas can only be adsorbed in the larger pores of moist coal, and provides an explanation for water weakening methane adsorption capacity.

Vessel co-option and angiotropic extravascular migratory metastasis: a continuum of tumour growth and spread?

Two fields of cancer research have emerged dealing with the biology of tumour cells localised to the abluminal vascular surface: vessel co-option (VCo), a non-angiogenic mode of tumour growth and angiotropic extravascular migratory metastasis (EVMM), a non-hematogenous mode of tumour migration and metastasis. VCo is a mechanism by which tumour cells gain access to a blood supply by spreading along existing blood vessels in order to grow locally. Angiotropic EVMM involves "pericytic mimicry" (PM), which is characterised by tumour cells continuously migrating in the place of pericytes distantly along abluminal vascular surfaces. When cancer cells are engaged in PM and EVMM, they migrate along blood vessels beyond the advancing front of the tumour to secondary sites with the formation of regional and distant metastases. In the present perspective, the authors review the current scientific literature, emphasising the analogies between embryogenesis and cancer progression, the re-activation of embryonic signals by "cancer stem cells", and the important role of laminins and epithelial-mesenchymal-transition. This perspective maintains that VCo and angiotropic EVMM constitute complementary processes and represent a continuum of cancer progression from the primary tumour to metastases and of tumour growth to EVMM, analogous to the embryonic development program.

A need for stratification of metastasis samples according to secondary site in gene expression studies

Comparisons of gene expression profiles between primary tumors and metastasis have revealed genes that are implicated in metastasis formation. However, gene expression studies conducted on metastasis samples from the same primary site usually do not discriminate between different secondary sites. Although the change in the expression of number of genes is expected to be common to metastasis from the same primary but different secondary sites, herein the data that point to substantial differences are presented. Furthermore, the reciprocal communication between metastatic and host cells that is influencing these differences is outlined to emphasize the need for stratification of metastasis samples in gene expression studies.

Vegetation of temperate inland salt marshes on their north-western border (North German Plain)

Temperate inland salt marshes, formerly used as meadows or pastures, are priority habitats in Europe and are threatened by intensifying anthropogenic activites. They are particularly important because of their biogeographical location on the North German Plain, which is the westernmost hotspot of continental halophytic vegetation in Eurasia. In spite of their remarkably long history of floristic research, there are disproportionately few studies dealing with plant communities. They are traditionally included in the Puccinellion maritimae and Armerion maritimae alliances with a typical distribution along the North and Baltic Sea coasts. The similarity with other inland salt marshes has been overlooked. We surveyed natural and secondary sites with euhalophytic vegetation in central Germany (Saxony-Anhalt and Thuringia) in 2018 and 2020 and analysed 105 phytosociological relevés. This resulted in the identification of three main groups (alliances) containing seven associations: annual hypersaline succulent communities of the alliance Therosalicornion (Salicornietum europaeae, Halimioni pedunculatae-Puccinellietum distantis, Suaedetum maritimae), wet saline meadows of the alliance Juncion gerardi (Triglochino maritimae-Glaucetum maritimae, Scorzonero parviflorae-Juncetum gerardi variant with Glaux maritima) and saline short-grass swards of the alliance Puccinellion limosae (Puccinellietum limosae, Atriplex prostrata community). For each association we defined the typical species composition and calculated Ellenberg indicator values depending on the ecological gradients of soil salinity, nutrients and moisture. To determine possible floristic similarities between the inland and coastal salt marshes, we compared our data with data from other areas of saline vegetation: the southern Baltic Sea coast, Kujawy (Poland), Pannonian Basin (Hungary) and Transylvanian Basin (Romania). We concluded that the salt marshes in inland Germany showed a stronger relation with salt marshes of the remote Pannonian and Transylvanian Basins, and they were well separated from the Baltic salt marshes. Thus, we suggest including inland salt marshes on the North German Plain into the syntaxonomical class of continental halophytic vegetation of Festuco-Puccinellietea.

Vascular inflammation in the central nervous system.

Vascular inflammation in the central nervous system.

Patient-derived xenograft models of neuroendocrine prostate cancer.

In recent years, patient-derived xenografts (PDXs) have attracted much attention as clinically relevant models for basic and translational cancer research. PDXs retain the principal histopathological and molecular heterogeneity of their donor tumors and remain stable across passages. These characteristics allow PDXs to offer a reliable platform for better understanding cancer biology, discovering biomarkers and therapeutic targets, and developing novel therapies. A growing interest in generating neuroendocrine prostate cancer (NEPC) PDX models has been demonstrated, and such models have proven useful in several areas. This review provides a comprehensive summary of currently available NEPC PDX collections, encompassing 1) primary or secondary sites where patient samples were collected, 2) donor patients' treatment histories, 3) morphological features (i.e., small cell and large cell), and 4) genomic alterations. We also highlight suitable models for various research purposes, including identifying therapeutic targets and evaluating drug responses in models with specific genomic backgrounds. Finally, we provide perspectives on the current knowledge gaps and shed light on future applications and improvements of NEPC PDXs.

Repression of Cell-to-Matrix Adhesion by Metformin Chloride Supports Its Anti-Metastatic Potential in an In Vitro Study on Metastatic and Non-Metastatic Cancer Cells.

Glucose metabolism has significant impact on cancer cell survival and proliferation. Our previous studies have shown that level of glucose in the medium affects the cell attachment to and detachment from the substratum. Control of glucose metabolism in cancer cells has potential to serve as an anti-cancer therapy. Different anti-diabetic drugs have been reported to inhibit glucose uptake at cellular level by glucose transporters. Metformin chloride is commonly used as antidiabetic drug. It is known that use of metformin reduces chances of developing cancer in diabetic patients. Here we have investigated the effect of metformin on cell adhesion proteins and other related factors in different cancer cells, both metastatic and non-metastatic. The object was to evaluate the effect of different doses of metformin on the onset of metastasis after these are detached from their primary site of origin and re-attached at the secondary site. For this purpose, we grew different cancer cells (MDA-MB231, MCF7, HCT116, and SF767) in culturing media containing different concentrations of metformin chloride. Quantitative real-time PCR was used to evaluate the expression profile of the genes involved in cell adhesion. It was observed that metformin treatment increased the expression of epithelial isoforms of cell adhesion molecules along with integrins responsible for cell-to-matrix adhesion and inducing specific morphological changes such as development of cytoskeletal structures in different cancer cell lines which normally lead to attainment of mesenchymal phenotype. The effect of metformin appeared to be different in different doses. The glioblastoma cells (SF767) were observed to be the most sensitive cells among all cancer cells under study. Our data supports the idea that the metformin prevents the cancer cells from acquiring mesenchymal phenotype and hence prevents onset of metastasis, and if the process has already started then it has potential to prevent cancer cell attachment to the secondary site.

Epidemic containment and social control measures in Portugal: the line of lazarettos protecting the land border (1884–1886)

Despite the frequent epidemics that affected the Iberian Peninsula, Portugal only set up a system of permanent lazarettos along its border with Spain in the late nineteenth century, during the cholera outbreaks of 1884–1886. Planned and rolled out by army doctors, the system consisted of five primary sites (located at rail crossing points and river boat stations that linked the two sides of the border) and smaller, secondary sites (“vigilance and isolation posts”) at every railway station in the country. This contribution will focus especially on the epidemic containment measures (observation, isolation, and treatment of the infected) implemented in the lazarettos and at the vigilance posts, most of which were refitted and turned into virtual high-security prisons under military guard after the 1884 cholera outbreak. It will also examine the interaction between these preventive measures, which were also aimed at social control and control of unauthorised movement, and the land border cordons sanitaires and the sanitary inspection posts and quarantine complexes that protected the seaports, which functioned as a permanent maritime cordon sanitaire along the Portuguese coast.

Topical Autologous Blood Clot Therapy: An Introduction and Development of Consensus Panel to Guide Use in the Treatment of Complex Wound Types

Complex or hard-to-heal wounds can be acute or chronic; the complexity is based on patient-specific local, systemic, and psychosocial factors. Use of autologous tissue can be a significant adjunct to wound closure. Grafts and flaps are the most common autologous tissue used in wound reconstruction. However, patient factors, wound size, and exposed structures may preclude using these methods as primary or even secondary closure techniques. Alternative autologous tissue therapies include those derived from adipose, epidermis or dermis, bone marrow, and blood. Limitations of these treatment modalities include access-related difficulty, cost, creation of a secondary donor site, use of singular or limited cell types, and sparse or contradictory evidence basis of their efficacy of use. A panel of providers experienced in wound care and surgical wound management was convened to create a series of publications on the use of topical autologous blood clot therapy (TABCT) in the treatment of complex wounds. This publication, the first in a series, provides an evidence basis of the gap between definition and treatment of complex wounds, an overview of the use of autologous therapies in these wounds, and the science behind TABCT. The development of a consensus panel for decision pathways and recommendations for TABCT use in specific complex wound types are also discussed. Subsequent articles will provide consensus recommendations on the use of TABCT in full-thickness wounds with exposed tendon and/or bone and undermining or tunneling wounds, in wounds in patients who are nonsurgical candidates, in those who cannot undergo sharp debridement, in patients with arterial wounds who have been maximally revascularized, and in those with transsphincteric anal fistula. This article provides a foundation of knowledge and describes the plan for consensus panel decision pathways and recommendation development of use of TABCT in the treatment of specific complex wound types.

Complementarity of the residue-level protein function and structure predictions in human proteins

Sequence-based predictors of the residue-level protein function and structure cover a broad spectrum of characteristics including intrinsic disorder, secondary structure, solvent accessibility and binding to nucleic acids. They were catalogued and evaluated in numerous surveys and assessments. However, methods focusing on a given characteristic are studied separately from predictors of other characteristics, while they are typically used on the same proteins. We fill this void by studying complementarity of a representative collection of methods that target different predictions using a large, taxonomically consistent, and low similarity dataset of human proteins. First, we bridge the gap between the communities that develop structure-trained vs. disorder-trained predictors of binding residues. Motivated by a recent study of the protein-binding residue predictions, we empirically find that combining the structure-trained and disorder-trained predictors of the DNA-binding and RNA-binding residues leads to substantial improvements in predictive quality. Second, we investigate whether diverse predictors generate results that accurately reproduce relations between secondary structure, solvent accessibility, interaction sites, and intrinsic disorder that are present in the experimental data. Our empirical analysis concludes that predictions accurately reflect all combinations of these relations. Altogether, this study provides unique insights that support combining results produced by diverse residue-level predictors of protein function and structure.

Desialylation of O‐glycans activates von Willebrand factor by destabilizing its autoinhibitory module

BackgroundThe binding of the A1 domain of von Willebrand factor (VWF) to platelet receptor glycoprotein (GP)Ibα defines the VWF activity in hemostasis. Recent studies suggest that sequences flanking A1 form cooperatively an autoinhibitory module (AIM) that reduces the accessibility of the GPIbα binding site on A1. Application of a tensile force induces unfolding of the AIM. Desialylation induces spontaneous binding of plasma VWF to platelets. Most O‐glycans in VWF are located around the A1 domain. Removing certain O‐glycans in the flanking sequences by site‐directed mutagenesis enhances A1 binding to GPIbα and produces an effect similar to type 2B von Willebrand disease in animals. ObjectivesTo understand if and how desialylation of O‐glycans in the flanking sequences increases A1 activity. MethodsA recombinant AIM‐A1 fragment encompassing VWF residues 1238–1493 and only O‐glycans was treated with neuraminidase to produce desialylated protein. The glycan structure, dynamics, stability, and function of the desialylated protein was characterized by biochemical and biophysical methods and compared to the sialylated fragment. ResultsAsialo‐AIM‐A1 exhibited increased binding activity and induced more apparent platelet aggregation than its sialylated counterpart. It exhibited a lower melting temperature, and increased hydrogen–deuterium exchange rates at residues near the secondary GPIbα binding site and the N‐terminal flanking sequence. Asialo‐AIM‐A1 is less mechanically stable than sialo‐AIM‐A1, with its unstressed unfolding rate approximately 3‐fold greater than the latter. ConclusionsDesialylation of O‐glycans around A1 increases its activity by destabilizing the AIM.

Delivery of an immunogenic cell death-inducing copper complex to cancer stem cells using polymeric nanoparticles.

The major cause for cancer related deaths worldwide is tumour relapse and metastasis, both of which have been heavily linked to the existence of cancer stem cells (CSCs). CSCs are able to escape current treatment regimens, reform tumours, and promote their spread to secondary sites. Recently, our research group reported the first metal-based agent 1 (a copper(ii) compound ligated by a bidentate 4,7-diphenyl-1,10-phenanthroline and a tridentate Schiff base ligand) to potently kill CSCs via cytotoxic and immunogenic mechanisms. Here we show that encapsulation of 1 by polymeric nanoparticles at the appropriate feed (10%, 1 NP10) enhances CSC uptake and improves potency towards bulk cancer cells and CSCs (grown in monolayer and three-dimensional cultures). The nanoparticle formulation triggers a similar cellular response to the payload, which bodes well for further translation. Specifically, the nanoparticle formulation elevates intracellular reactive oxygen species levels, induces ER stress, and evokes damage-associated molecular patterns consistent with immunogenic cell death. To the best of our knowledge, this is the first study to demonstrate that polymeric nanoparticles can be used to effectively deliver immunogenic metal complexes into CSCs.

Targeting Oxidative Phosphorylation-Proteasome Activity in Extracellular Detached Cells Promotes Anoikis and Inhibits Metastasis.

Metastasis arises owing to tumor cells’ capacity to evade pro-apoptotic signals. Anoikis—the apoptosis of detached cells (from the extracellular matrix (ECM)) is often circumvented by metastatic cells as a result of biochemical and molecular transformations. These facilitate cells’ ability to survive, invade and reattach to secondary sites. Here, we identified deregulated glucose metabolism, oxidative phosphorylation, and proteasome in anchorage-independent cells compared to adherent cells. Metformin an anti-diabetic drug that reduces blood glucose (also known to inhibit mitochondrial Complex I), and proteasome inhibitors were employed to target these changes. Metformin or proteasome inhibitors alone increased misfolded protein accumulation, sensitized tumor cells to anoikis, and impaired pulmonary metastasis in the B16F10 melanoma model. Mechanistically, metformin reduced cellular ATP production, activated AMPK to foster pro-apoptotic unfolded protein response (UPR) through enhanced expression of CHOP in ECM detached cells. Furthermore, AMPK inhibition reduced misfolded protein accumulation, thus highlight relevance of AMPK activation in facilitating metformin-induced stress and UPR cell death. Our findings provide insights into the molecular biology of anoikis resistance and identified metformin and proteasome inhibitors as potential therapeutic options for tumor metastasis.

Direct H abstraction by molecular oxygen from unsaturated C3–C5 hydrocarbons: A theoretical study

AbstractPotential energy surfaces for the H abstraction reactions by molecular oxygen from unsaturated closed‐shell C3–C5 hydrocarbons, including allene and propyne (C3H4), propene (C3H6), diacetylene (C4H2), vinylacetylene (C4H4), 1,3‐butadiene (C4H6), butene isomers (C4H8), cyclopentadiene (C5H6), and cyclopentene (C5H8), which were earlier identified among the main pyrolysis products of jet fuel components, have been explored at the CCSD(T)‐F12/cc‐pVTZ‐f12//ωB97XD/6‐311G** + ZPE(ωB97XD/6‐311G**) level of electronic structure theory. The results of the ab initio calculations were then utilized in transition state theory calculations of the reaction rate constants. The H abstraction reactions by O2 appeared to be most favorable from the allylic sites and from allene and propyne forming the propargyl radical, with the reactions involving the cyclic allylic sites in cyclopentadiene and cyclopentene exhibiting the lowest reaction endothermicities and barriers. They are followed by the reaction of O2 with cyclopentene producing cyclopent‐1‐en‐4‐yl and the reactions with vinylacetylene and 1,3‐butadiene forming the resonantly stabilized i‐C4H3 and i‐C4H5 radical products. The H abstractions from primary and secondary vinylic sites as well as from acetylenic sites are significantly less favorable and unlikely to compete. The calculated rate constants have been validated against the previous experimental and theoretical results for propene and butene isomers. All considered reactions of H abstraction by O2 are predicted to be rather slow, with their rate constants at 1500 K being on the order of 10–17‐10–16 cm3 molecule–1 s–1 and exhibiting a well‐defined Arrhenius behavior. Modified Arrhenius expressions for the reaction rate constants both in forward and reverse directions and Evans–Polanyi relationship between the activation and reaction energies have been generated and proposed for kinetic combustion models. The H abstractions by O2 forming the resonantly stabilized radicals were concluded to likely contribute to the initiation of oxidation of the unsaturated C3–C5 species produced in the pyrolysis of the important components of jet fuels.

Targeted siRNA Nanotherapeutics Against Breast and Ovarian Metastatic Cancer: A Comprehensive Review of the Literature

Metastasis is considered the major cause of unsuccessful cancer therapy. The metastatic development requires tumor cells to leave their initial site, circulate in the blood stream, acclimate to new cellular environments at a remote secondary site and endure there. There are several steps in metastasis, including invasion, intravasation, circulation, extravasation, premetastatic niche formation, micrometastasis and metastatic colonization. siRNA therapeutics are appreciated for their usefulness in treatment of cancer metastasis. However, siRNA therapy as a single therapy may not be a sufficient option for control of metastasis. By combining siRNA with targeting, functional agents or small-molecule drugs have shown potential effects that enhance therapeutic effectiveness. This review addresses multidrug resistance and metastasis in breast and ovarian cancers and highlights drug-delivery strategies using siRNA therapeutics.