Membrane trafficking alterations in breast cancer progression.

Breast cancer (BC) is the most common type of cancer in women, and remains one of the major causes of death in women worldwide. It is now well established that alterations in membrane trafficking are implicated in BC progression. Indeed, membrane trafficking pathways regulate BC cell proliferation, migration, invasion, and metastasis. The 22 members of the ADP-ribosylation factor (ARF) and the >60 members of the rat sarcoma (RAS)-related in brain (RAB) families of small GTP-binding proteins (GTPases), which belong to the RAS superfamily, are master regulators of membrane trafficking pathways. ARF-like (ARL) subfamily members are involved in various processes, including vesicle budding and cargo selection. Moreover, ARFs regulate cytoskeleton organization and signal transduction. RABs are key regulators of all steps of membrane trafficking. Interestingly, the activity and/or expression of some of these proteins is found dysregulated in BC. Here, we review how the processes regulated by ARFs and RABs are subverted in BC, including secretion/exocytosis, endocytosis/recycling, autophagy/lysosome trafficking, cytoskeleton dynamics, integrin-mediated signaling, among others. Thus, we provide a comprehensive overview of the roles played by ARF and RAB family members, as well as their regulators in BC progression, aiming to lay the foundation for future research in this field. This research should focus on further dissecting the molecular mechanisms regulated by ARFs and RABs that are subverted in BC, and exploring their use as therapeutic targets or prognostic markers.

The artoriine wolf spiders of Australia: the new genus Kochosa and a key to genera (Araneae: Lycosidae).

A key to the six Australian genera of the wolf spider (Lycosidae Sundevall, 1833) subfamily Artoriinae Framenau, 2007 is provided, now including Artoria Thorell, 1877, Artoriopsis Framenau, 2007, Diahogna Roewer, 1960, Kangarosa Framenau, 2010, Kochosa gen. nov. and Tetralycosa Roewer, 1960. Kochosa gen. nov. is described to include 16 species: K. australia sp. nov. (type species; from New South Wales, Queensland, South Australia, Victoria, Western Australia), K. aero sp. nov. (Western Australia), K. asterix sp. nov. (New South Wales, Queensland, Tasmania and Victoria), K. confusa sp. nov. (Queensland), K. erratum sp. nov. (Queensland), K. fleurae sp. nov. (Victoria), K. mendum sp. nov. (Australian Capital Territory, New South Wales, Queensland), K. nigra sp. nov. (Queensland), K. obelix sp. nov. (Western Australia), K. queenslandica sp. nov. (Queensland), K. sharae sp. nov. (South Australia), K. tanakai sp. nov. (New South Wales, Queensland), K. tasmaniensis sp. nov. (Tasmania), K. timwintoni sp. nov. (Western Australia), K. tongiorgii sp. nov., (Queensland), and K. westralia sp. nov. (Western Australia). Kochosa gen. nov. differs distinctly from all other genera within the Artoriinae by somatic and genitalic morphology. Most conspicuous is a distinct off-white or yellowish-white cardiac mark on an otherwise generally uniformly dark abdomen. The cardiac mark is rendered by thick black setae, which are particularly dense posteriorly. The tegular apophysis of the male pedipalp is heavily reduced, generally forming a semi-transparent small lobe. In turn, the embolic division is often complex with a variety of apophyses. Kochosa gen. nov. generally inhabit mesic habitats such as temperate and tropical shrubs and forests along the eastern and south-eastern coast and in the south-western parts of Australia.

Expression in Escherichia coli, Refolding, and Purification of Plant Aspartic Proteases.

Aspartic proteases (APs) are widely distributed in plants. The large majority of genes encoding putative APs exhibit distinct features when compared with the so-called typical APs, and have been grouped as atypical and nucellin-like APs. Remarkably, a diverse pattern of enzymatic properties, subcellular localizations, and biological functions are emerging for these proteases, illustrating the functional complexity among plant pepsin-like proteases. However, many key questions regarding the structure-function relationships of plant APs remain unanswered. Therefore, the expression of these enzymes in heterologous systems is a valuable strategy to unfold the unique features/biochemical properties among members of this family of proteases. Here, we describe our protocol for the production and purification of recombinant plant APs, using a procedure where the protein is refolded from inclusion bodies by dialysis. This method allows the production of untagged versions of the target protease, which has revealed to be critical to disclose differences in processing/activation requirements between plant APs. The protocol includes protein expression, washing and solubilization of inclusion bodies, refolding by dialysis, and a protein purification method. Specific considerations on critical aspects of the refolding process and further suggestions for evaluation of the final recombinant product are also provided.

Two new species in the orb-weaving spider genus Larinia Simon, 1874 (Araneae, Araneidae) from Western Australia.

Two new species in the orb-weaving spider genus Larinia Simon, 1874 are described, L. sexta n. sp. and L. tumulus n. sp. This elevates the Australian number of described species in the genus to seven. With the exception of two females of L. sexta n. sp. recorded from mainland Western Australia, both species have so far exclusively been found on Barrow Island, 50 km off the north-western Western Australian coast where a third species, L. montagui Hogg, 1914, also occurs. Both new species appear to favour spinifex (Triodia spp.) grassland, but specimen numbers in collections are too low to accurately characterize life history patterns and habitat preferences.

Revision of the new Australasian orb-weaving spider genus Salsa (Araneae, Araneidae).

A new Australasian genus in the orb-weaving spider family Araneidae Clerck, 1757 is described to include seven species: Salsafuliginata (L. Koch, 1871) comb. nov. (type species; = Epeirarubicundula Keyserling, 1887) syn. nov.) (Australia, introduced to New Zealand); S.brisbanae (L. Koch, 1867) comb. nov. (Australia); S.canalae (Berland, 1924) comb. nov. (New Caledonia); S.neneba sp. nov. (Papua New Guinea); S.recherchensis (Main, 1954) comb. nov. (Australia); S.rueda sp. nov. (Australia); and S.tartara sp. nov. (Australia; Lord Howe Island endemic). Salsa gen. nov. belongs to the Australasian informal backobourkiine clade and differs from other genera of this clade by a distinct abdominal shape (single posterior abdominal tubercle) and ventral colouration (pale lateral spindle-shaped bands), male pedipalp morphology (C-shaped median apophysis that has teeth-like tubercles inside the basal arch) and the shape of the female epigyne scape (partially translucent and generally shorter than the epigyne plate). Based mainly on male pedipalp morphology within the backobourkiines, Salsa gen. nov. has closest morphological affinities with Acroaspis Karsch, 1878 and Socca Framenau, Castanheira & Vink, 2022.

Implications of a cheliceral axial duplication in Tetragnatha versicolor (Araneae: Tetragnathidae) for arachnid deuterocerebral appendage development.

The homology of the arachnid chelicera with respect to other head appendages in Panarthropoda has long been debated. Gene expression data and the re-interpretation of early transitional fossils have supported the homology of the deutocerebrum and its associated appendages, implying a homology between primary antennae (mandibulates), chelicerae (euchelicerates), and chelifores (sea spiders). Nevertheless, comparatively little is known about the mechanistic basis of proximo-distal (PD) axis induction in chelicerates, much less the basis for cheliceral fate specification. Here, we describe a new cheliceral teratology in the spider Tetragnatha versicolor Walckenaer, 1841, which consists on a duplication of the PD axis of the left chelicera associated with a terminal secondary schistomely on the fang of the lower axis. This duplication offers clues as to potential shared mechanisms of PD axis formation in the chelicera. We review the state of knowledge on PD axis induction mechanisms in arthropods and identify elements of gene regulatory networks that are key for future functional experiments of appendage development in non-insect model systems. Such investigations would allow a better understanding of PD axis induction of modified and poorly studied arthropod limbs (e.g., chelicerae, chelifores, and ovigers).

Notes on slender species of the long-jawed spider genus Tetragnatha (Araneae, Tetragnathidae) with description of three new species.

New data about slender orb-weaving species of the cosmopolitan genus Tetragnatha are presented. Tetragnatha chauliodus (Thorell, 1890) and Tetragnatha tenuissima O. Pickard-Cambridge, 1889 are redescribed, including one synonymy for each species and the first record of the first species to the Neotropical region. Also, three new species are herein described, all based on males and females. Tetragnatha megalocera new species is recorded exclusively from Brazil (Espírito Santo, Rio de Janeiro, Rio Grande do Sul and São Paulo states), while Tetragnatha renatoi new species is recorded from Venezuela, Argentina (Misiones) and Brazil (Paraná, Pernambuco, Rio Grande do Sul, Rondônia, Santa Catarina and São Paulo states). Finally, Tetragnatha chiyokoae new species is described from Yunnan province (China) and Okinawa (Japan), with an additional record for Taiwan. Furthermore, Tetragnatha exilima (Mello-Leitão, 1943), Tetragnatha filigastra Mello-Leitão, 1943 and Tetragnatha lactescens (Mello-Leitão, 1947) are nomina dubia.

Three new species of Ochyrocera (Araneae, Ochyroceratidae) from Southeastern Brazil.

Three new species of the six-eyed  haplogyne and ecribellate spiders from the genus Ochyrocera Simon, 1891 are illustrated and described based on males and females from southeastern Brazil: Ochyrocera tinocoi new species (Sooretama, Espírito Santo state), Ochyrocera garayae new species (Linhares and Sooretama, Espírito Santo state) and Ochyrocera itatinga new species (Rio de Janeiro city, Rio de Janeiro state). The new species expand the distribution range of Ochyrocera in Brazil and increase to 50 the total number of species described, from which hitherto 33 species occur in South America, acknowledging the high diversity of the genus for the region.

Arl13b Regulates Breast Cancer Cell Migration and Invasion by Controlling Integrin-Mediated Signaling.

Breast cancer is the first cause of cancer-related mortality among women worldwide, according to the most recent estimates. This mortality is mainly caused by the tumors' ability to form metastases. Cancer cell migration and invasion are essential for metastasis and rely on the interplay between actin cytoskeleton remodeling and cell adhesion. Therefore, understanding the mechanisms by which cancer cell invasion is controlled may provide new strategies to impair cancer progression. We investigated the role of the ADP-ribosylation factor (Arf)-like (Arl) protein Arl13b in breast cancer cell migration and invasion in vitro, using breast cancer cell lines and in vivo, using mouse orthotopic models. We show that Arl13b silencing inhibits breast cancer cell migration and invasion in vitro, as well as cancer progression in vivo. We also observed that Arl13b is upregulated in breast cancer cell lines and patient tissue samples. Moreover, we found that Arl13b localizes to focal adhesions (FAs) and interacts with ß3-integrin. Upon Arl13b silencing, ß3-integrin cell surface levels and FA size are increased and integrin-mediated signaling is inhibited. Therefore, we uncover a role for Arl13b in breast cancer cell migration and invasion and provide a new mechanism for how ARL13B can function as an oncogene, through the modulation of integrin-mediated signaling.

Use of recombinant proteins as a simple and robust normalization method for untargeted proteomics screening: exhaustive performance assessment.

The label-free quantitative mass spectrometry methods, in particular, the SWATH-MS approach, have gained popularity and became a powerful technique for comparison of large datasets. In the present work, it is evaluated the use of recombinant proteins as internal standards for untargeted label-free methods. The proposed internal standard strategy reveals a similar intragroup normalization capacity when compared with the most common normalization methods, with the additional advantage of maintaining the overall proteome changes between groups (which is buffered with the use of other methods). Therefore, the proposed strategy is able to maintain a good performance even when large qualitative and quantitative differences in sample composition are observed, such as the ones induced by biological regulation (as observed in secretome and other biofluids' analyses) or by enrichment approaches (such as immunopurifications). Moreover, this approach corresponds to a cost-effective and simple normalization method altrenative, therefore being an appealing strategy for large quantitative screening, as the analysis of clinical cohorts for biomarker discovery.

oxSWATH: An integrative method for a comprehensive redox-centered analysis combined with a generic differential proteomics screening.

Most of the redox proteomics strategies are focused on the identification and relative quantification of cysteine oxidation without considering the variation in the total levels of the proteins. However, protein synthesis and protein degradation also belong to the regulatory mechanisms of the cells, being therefore important to consider the changes in total protein levels in PTMs-focused analyses, such as cysteine redox characterization. Therefore, a novel integrative approach combining the SWATH-MS method with differential alkylation using a combination of commonly available alkylating reagents (oxSWATH) is presented, by which it is possible to integrate the information regarding relative cysteine oxidation with the analysis of the total protein levels in a cost-effective high-throughput approach. The proposed method was tested using a redox-regulated protein and further applied to a comparative analysis of secretomes obtained from cells cultured under control or oxidative stress conditions to strengthen the importance of considering the overall proteome changes. Using the OxSWATH method it was possible to determine both the relative proportion of reduced and reversible oxidized oxoforms, as well as the total levels of each oxoform by taking into consideration the total levels of the protein. Therefore, using OxSWATH the comparative analyses can be performed at two different levels by considering the relative proportion or the total levels at both peptide and protein level. Moreover, since samples are acquired in SWATH-MS mode, besides the redox centered analysis, a generic differential protein expression analysis can also be performed, allowing a truly comprehensive evaluation of proteomics changes upon the oxidative stimulus. Data are available via ProteomeXchange and SWATHAtlas with the identifiers PXD006802, PXD006802, and PASS01210.

Cardosin A endocytosis mediated by integrin leads to lysosome leakage and apoptosis of epithelial cells.

Cardosin A is an aspartic protease present in large amount in the pistils of cardoon flowers. This protease is known to contain an -Arg-Gly-Asp- (RGD) motif located on the molecular surface. In this study, we found that isolated recombinant cardosin A attached to human epithelial cells A549, mediated by the binding of its RGD motif to cell surface integrins. The cell bound cardosin A was internalized to endosomes and lysosomes and triggered the permeability of lysosomal membrane leading to apoptosis of the epithelial cells. These events are identical to those observed for three RGD-containing aspartic proteases, Saps 4-6, secreted by Candida albicans. Such a process, which has been called the Trojan Horse mechanism, is believed to benefit the invasion of C. albican into the epithelium of the host. The location of the RGD motifs of cardosin A and Saps 4-6 are on the opposite ends of the homologous three-dimensional structures, suggesting that the Trojan Horse mechanism is insensitive to the RGD position. Current finding also suggests that cardosin A may have a defensive function against the ingestion of cardoon flowers by human, insects, and other herbivores.

The scorpion-tailed orb-weaving spiders (Araneae, Araneidae, Arachnura) in Australia and New Zealand.

The scorpion-tailed orb-weaving spiders in the genus Arachnura Vinson, 1863 (Araneidae Clerck, 1757) are revised for Australia and New Zealand. Arachnura higginsii (L. Koch, 1872) only occurs in Australia and A. feredayi (L. Koch, 1872) only in New Zealand. A single female collected in south-eastern Queensland (Australia) is here tentatively identified as A. melanura Simon, 1867, but it is doubtful that this species has established in Australia. Two juveniles from northern Queensland do not conform to the diagnoses of any of the above species and are illustrated pending a more thorough revision of the genus in South-East Asia and the Pacific region. An unidentified female from Westport (New Zealand) does not conform to the diagnoses of A. feredayi and A. higginsii, but is not described due to its poor preservation status. Arachnura caudatella Roewer, 1942 (replacement name for Epeira caudata Bradley, 1876), originally described from Hall Sound (Papua New Guinea) and repeatedly catalogued for Australia, is considered a nomen dubium.

Notes on the orb-weaving spider genus Alpaida (Araneae, Araneidae) with description of four new species from Rio de Janeiro, Brazil.

Four new species of the orb-weaving spider genus Alpaida O. P.-Cambridge, 1889 from Rio de Janeiro state, Brazil are illustrated and described based on males and females from the following municipalities: Alpaida imperatrix new species (Macaé and Rio de Janeiro); Alpaida imperialis new species (Mendes and Rio de Janeiro); Alpaida marista new species (Mendes and Pinheiral); and Alpaida mendensis new species (Mendes). Furthermore, two new synonymies are herein proposed: Alpaida lanei Levi, 1988 = Alpaida atomaria (Simon, 1895) and Alpaida caxias Levi, 1988 = Alpaida tijuca Levi, 1988, alongside new records for both species and also Alpaida venger Castanheira Baptista, 2015.

Spider diversity (Arachnida: Araneae) in Atlantic Forest areas at Pedra Branca State Park, Rio de Janeiro, Brazil.

BACKGROUND: There has never been any published work about the diversity of spiders in the city of Rio de Janeiro using analytical tools to measure diversity. The only available records for spider communities in nearby areas indicate 308 species in the National Park of Tijuca and 159 species in Marapendi Municipal Park. These numbers are based on a rapid survey and on an one-year survey respectively. NEW INFORMATION: This study provides a more thorough understanding of how the spider species are distributed at Pedra Branca State Park. We report a total of 14,626 spider specimens recorded from this park, representing 49 families and 373 species or morphospecies, including at least 73 undescribed species. Also, the distribution range of 45 species was expanded, and species accumulation curves estimate that there is a minimum of 388 (Bootstrap) and a maximum of 468 species (Jackknife2) for the sampled areas. These estimates indicates that the spider diversity may be higher than observed.

Biophysical characterization of laforin-carbohydrate interaction.

Laforin is a human dual-specificity phosphatase (DSP) involved in glycogen metabolism regulation containing a carbohydrate-binding module (CBM). Mutations in the gene coding for laforin are responsible for the development of Lafora disease, a progressive fatal myoclonus epilepsy with early onset, characterized by the intracellular deposition of abnormally branched, hyperphosphorylated insoluble glycogen-like polymers, called Lafora bodies. Despite the known importance of the CBM domain of laforin in the regulation of glycogen metabolism, the molecular mechanism of laforin-glycogen interaction is still poorly understood. Recently, the structure of laforin with bound maltohexaose was determined and despite the importance of such breakthrough, some molecular interaction details remained missing. We herein report a thorough biophysical characterization of laforin-carbohydrate interaction using soluble glycans. We demonstrated an increased preference of laforin for the interaction with glycans with higher order of polymerization and confirmed the importance of tryptophan residues for glycan interaction. Moreover, and in line with what has been described for other CBMs and lectins, our results confirmed that laforin-glycan interactions occur with a favourable enthalpic contribution counter-balanced by an unfavourable entropic contribution. The analysis of laforin-glycan interaction through the glycan side by saturation transfer difference (STD)-NMR has shown that the CBM-binding site can accommodate between 5 and 6 sugar units, which is in line with the recently obtained crystal structure of laforin. Overall, the work in the present study complements the structural characterization of laforin and sheds light on the molecular mechanism of laforin-glycan interaction, which is a pivotal requisite to understand the physiological and pathological roles of laforin.

Corrigendum: Unveiling the potential of novel yeast protein extracts in white wines clarification and stabilization.

[This corrects the article on p. 20 in vol. 3, PMID: 25853122.].

A look into amyloid formation by transthyretin: aggregation pathway and a novel kinetic model.

The aggregation of proteins into insoluble amyloid fibrils is the hallmark of many, highly debilitating, human pathologies such as Alzheimer's or Parkinson's disease. Transthyretin (TTR) is a homotetrameric protein implicated in several amyloidoses like Senile Systemic Amyloidosis (SSA), Familial Amyloid Polyneuropathy (FAP), Familial Amyloid Cardiomyopathy (FAC), and the rare Central Nervous System selective Amyloidosis (CNSA). In this work, we have investigated the kinetics of TTR aggregation into amyloid fibrils produced by the addition of NaCl to acid-unfolded TTR monomers and we propose a mathematically simple kinetic mechanism to analyse the aggregation kinetics of TTR. We have conducted circular dichroism, intrinsic tryptophan fluorescence and thioflavin-T emission experiments to follow the conformational changes accompanying amyloid formation at different TTR concentrations. Kinetic traces were adjusted to a two-step model with the first step being second-order and the second being unimolecular. The molecular species present in the pathway of TTR oligomerization were characterized by size exclusion chromatography coupled to multi-angle light scattering and by transmission electron microscopy. The results show the transient accumulation of oligomers composed of 6 to 10 monomers in agreement with reports suggesting that these oligomers may be the causative agent of cell toxicity. The results obtained may prove to be useful in understanding the mode of action of different compounds in preventing fibril formation and, therefore, in designing new drugs against TTR amyloidosis.

Dose-dependent inhibition of BACE-1 by the monoterpenoid 2,3,4,4-tetramethyl-5-methylenecyclopent-2-enone in cellular and mouse models of Alzheimer's disease.

BACE-1 is an aspartic protease involved in the conversion of amyloid precursor protein (APP) to amyloid-ß (Aß) in vivo, which is one of the key steps in the development and progression of Alzheimer's disease. In a previous screening procedure for inhibitors of BACE-1 activity, the oil of Lavandula luisieri was identified as the most potent among several essential oils. The inhibitory effect of this essential oil on Aß production was also demonstrated in a cellular assay. The composition of the volatile oil and the isolation of the compound responsible for the inhibitory activity were also reported. The present work focused on the characterization of the inhibition of BACE-1 by this active compound, a monoterpene necrodane ketone, 2,3,4,4-tetramethyl-5-methylenecyclopent-2-enone (1), with assessment of its Ki value and the type of inhibition. The dose-related effects of the compound were also evaluated using two different cell lines, with determinations of the respective EC50 values. The entire oil and the 2,3,4,4-tetramethyl-5-methylenecyclopent-2-enone (1) were tested on a triple transgenic mouse model of Alzheimer's disease. The overall results showed that compound 1 displayed a dose-dependent inhibition of BACE-1 in cellular and mouse models of Alzheimer's disease and is therefore capable of passing through cellular membranes and the blood-brain barrier.