Darwin's bark spider shares a spidroin repertoire with Caerostris extrusa but achieves extraordinary silk toughness through gene expression

Spider silk is a protein-based material whose toughness suggests possible novel applications. A particularly fascinating example of silk toughness is provided by Darwin's bark spider ( Caerostris darwini ) found in Madagascar. This spider produces extraordinarily tough silk, with an average toughness of 350 MJ m −1 and over 50% extensibility, and can build river-bridging webs with a size of 2.8 m 2 . Recent studies have suggested that specific spidroins expressed in C. darwini are responsible for the mechanical properties of its silk. Therefore, a more comprehensive investigation of spidroin sequences, silk thread protein contents and phylogenetic conservation among closely related species is required. Here, we conducted genomic, transcriptomic and proteomic analyses of C. darwini and its close relative Caerostris extrusa . A variety of spidroins and low-molecular-weight proteins were found in the dragline silk of these species; all of the genes encoding these proteins were conserved in both genomes, but their genes were more expressed in C. darwini . The potential to produce very tough silk is common in the genus Caerostris , and our results may suggest the existence of plasticity allowing silk mechanical properties to be changed by optimizing related gene expression in response to the environment.

Comparison of effectiveness of various bacterial proteases preparations in collagen hydrolysis

Гидролизаты коллагена широко используются в пищевой, фармацевтической и косметической промышленности. Для обработки коллагена используют различные протеолитические ферменты растительного, животного и микробного происхождения, из которых самыми перспективными являются бактериальные протеазы. Наиболее известные и часто используемые в пищевой отрасли коммерческие препараты бактериальных протеаз различаются по компонентному составу и могут содержать сериновые, металлопротеазы либо их смесь. С целью определения эффективности препаратов бактериальных протеаз с различным компонентным составом при получении гидролизатов коллагена мы провели ферментативную обработку говяжьего коллагена с использованием коммерческих препаратов Alcalase, Neutrase, Protamex и двух образцов ферментных препаратов, полученных в лабораторных условиях на основе новых отечественных штаммов Bacillus. Эффективность гидролиза оценивали по накоплению аминного азота, содержанию низкомолекулярного белка и по интенсивности белковых полос на электрофореграммах полученных продуктов. Все исследуемые препараты обеспечивали эффективный гидролиз коллагена. Наиболее интенсивно коллаген расщепляли препараты, содержащие только сериновые протеазы: Alcalase и ФП-145, полученный из культуральной жидкости штамма B. licheniformis-145. Высокую эффективность показали коммерческий препарат Protamex и ФП-96, полученный на основе мутантного варианта штамма B. subtilis-359 - продуцента субтилизина BPN’ и бациллолизина. Наименьший выход аминного азота и низкомолекулярных белков наблюдался в варианте с препаратом Neutrase, протеолитическая активность которого полностью представлена действием нейтральной протеазы бациллолизина. Лабораторные образцы ферментных препаратов, полученные на основе новых отечественных продуцентов, не уступали зарубежным коммерческим аналогам, а ФП-96 обеспечил более высокий выход аминного азота и низкомолекулярных белков в сравнении с широко используемым в пищевой промышленности препаратом Protamex. Collagen hydrolysates are widely used in the food, pharmaceutical and cosmetic industries. For collagen hydrolysis, various proteolytic enzymes of plant, animal and microbial origin are used, of which the most promising are bacterial proteases. The most well-known and commonly used in the food industry commercial preparations of bacterial proteases differ in their component composition and may contain serine protease, metalloproteases, or their mixture. In order to determine the effectiveness of bacterial protease preparations with different component composition in obtaining collagen hydrolysates, we carried out hydrolysis of beef collagen using commercial preparations Alcalase, Neutrase, Protamex and two laboratory samples of enzyme preparations obtained from new domestic Bacillus strains. The efficiency of hydrolysis was assessed by amine nitrogen accumulation, low-molecular-weight protein content, and by the intensity of the protein bands on the electrophoregrams of the hydrolysates. All investigated preparations provided effective collagen hydrolysis. Collagen was hydrolyzed most intensively by preparations containing only serine proteases: Alcalase and FP-145, obtained from B. licheniformis-145 culture liquid. The commercial preparation Protamex and FP-96, obtained from a mutant variant of the B. subtilis-359 strain, a producer of subtilisin BPN' and bacillolysin, showed high efficiency. The lowest yield of amine nitrogen and low molecular weight proteins was observed when using Neutrase, which contains only the neutral protease bacillolysin. Laboratory samples of enzyme preparations obtained from the new domestic producers were not inferior to foreign commercial counterparts, and FP-96 provided a higher yield of amine nitrogen and low molecular weight proteins in comparison with Protamex, which is widely used in the food industry.

Peculiarities of Fibrinolytic and Proteolytic Activity of Blood Plasma in Patients with Chronic Pancreatitis Combined with Hypothyroidism

The purpose of the study is to analyze the state of proteolytic and fibrinolytic activities of blood plasma in patients with chronic pancreatitis combined with hypothyroidism. Materials and methods. 105 people participated in our study, of which group 1 consisted of patients with chronic pancreatitis (n = 27), group 2 – patients with hypothyroidism (n = 30), group 3 – patients with chronic pancreatitis combined with hypothyroidism (n = 28), group 4 – almost healthy individuals (n = 20). The state of fibrinolytic activity of blood plasma was studied by lysis of azofibrin, followed by determination of total fibrinolytic activity, non-enzymatic fibrinolytic activity and enzymatic fibrinolytic activity. Assessment of the state of the proteolysis system was studied by lysis of azoalbumin (breakdown of low molecular weight proteins), azocasein (breakdown of high molecular weight proteins) and azocol (breakdown of collagen). Results. When analyzing the results of the study, we observe a probable increase in lysis of azoalbumin by 1.89, 1.96 and 2.16 times (p <0.05) in groups 1, 2, 3 compared with the group of almost healthy individuals. In patients with chronic pancreatitis and hypothyroidism, the most pronounced degradation of low molecular weight proteins was observed, which was 13.86% and 9.75% (p <0.05) higher than in the first and second groups. Indicators of azocasein lysis by 52.48%, 56.35% and 95.03% (p <0.05) were found in groups 1, 2, 3 compared with almost healthy individuals. Azocasein lysis was higher by 27.89% and 24.73% (p <0.05) in patients with chronic pancreatitis combined with hypothyroidism than in patients in groups 1 and 2. Azocol lysis was significantly higher by 10.85%, 12.05%, 16.87% (p <0.05) in groups 1, 2, 3 compared with almost healthy individuals. In addition, in patients with comorbid pathology there was an increase in lysis of azocol by 5.3% and 4.3% (p <0.05) compared with the first and second groups. The total fibrinolytic activity of blood plasma was 8.3%, 6.7%, 16.26% (p <0.05) lower in patients of groups 1, 2, 3 compared with almost healthy individuals. Non-enzymatic fibrinolytic activity of blood plasma was 44.89%, 49.64%, 66.27% higher in groups 1, 2 and 3 than in almost healthy individuals. Enzymatic fibrinolytic activity of blood plasma was 44.28%, 42.25%, 90.57% (p <0.05) lower in group 1, 2, 3 compared with the group of almost healthy individuals (p <0,05). There was a decrease in the level of enzymatic fibrinolytic activity of blood plasma by 32.07% and 33.96% (p <0.05) in patients with chronic pancreatitis associated with hypothyroidism compared with participants in groups 1 and 2 without comorbid pathology. Conclusion. The most pronounced changes in proteolytic (increased lysis of azoalbumin, azocasein, azocol) and fibrinolytic (decrease in total, non-enzymatic and enzymatic) activities of blood plasma in patients with chronic pancreatitis associated with hypothyroidism were determined

Darwin's bark spider shares a spidroin repertoire with Caerostris extrusa but achieves extraordinary silk toughness through gene expression

Spider silk is a protein-based material whose toughness suggests possible novel applications. A particularly fascinating example of silk toughness is provided by Darwin's bark spider (Caerostris darwini) found in Madagascar. This spider produces extraordinarily tough silk, with an average toughness of 350 MJ/m and over 50% extensibility, and can build river-bridging webs with a size of 2.8 m2. Recent studies have suggested that specific spidroins expressed in C. darwini are responsible for the mechanical properties of its silk. Therefore, a more comprehensive investigation of spidroin sequences, silk thread protein contents, and phylogenetic conservation among closely related species is required. Here, we conducted genomic, transcriptomic, and proteomic analyses of C. darwini and its close relative Caerostris extrusa. A variety of spidroins and low-molecular-weight proteins were found in the dragline silk of these species; all of the genes encoding these proteins were conserved in both genomes, but their genes were more expressed in C. darwini. The potential to produce very tough silk is common in the genus Caerostris, and our results may suggest the existence of plasticity allowing silk mechanical properties to be changed by optimizing related gene expression in response to the environment.

The (pro)renin receptor (ATP6ap2) facilitates receptor-mediated endocytosis and lysosomal function in the renal proximal tubule

The ATP6ap2 (Pro)renin receptor protein associates with H+-ATPases which regulate organellar, cellular, and systemic acid–base homeostasis. In the kidney, ATP6ap2 colocalizes with H+-ATPases in various cell types including the cells of the proximal tubule. There, H+-ATPases are involved in receptor-mediated endocytosis of low molecular weight proteins via the megalin/cubilin receptors. To study ATP6ap2 function in the proximal tubule, we used an inducible shRNA Atp6ap2 knockdown rat model (Kd) and an inducible kidney-specific Atp6ap2 knockout mouse model. Both animal lines showed higher proteinuria with elevated albumin, vitamin D binding protein, and procathepsin B in urine. Endocytosis of an injected fluid-phase marker (FITC- dextran, 10 kDa) was normal whereas processing of recombinant transferrin, a marker for receptor-mediated endocytosis, to lysosomes was delayed. While megalin and cubilin expression was unchanged, abundance of several subunits of the H+-ATPase involved in receptor-mediated endocytosis was reduced. Lysosomal integrity and H+-ATPase function are associated with mTOR signaling. In ATP6ap2, KO mice mTOR and phospho-mTOR appeared normal but increased abundance of the LC3-B subunit of the autophagosome was observed suggesting a more generalized impairment of lysosomal function in the absence of ATP6ap2. Hence, our data suggests a role for ATP6ap2 for proximal tubule function in the kidney with a defect in receptor-mediated endocytosis in mice and rats.

PHYLOGENETIC AFFINITY OF RAT AND SOME MAMMALIAN SPECIES METALLOTHIONEIN GENES

Metallothioneins ( are low molecular weight proteins, which have ability to bind bothmono and divalent metals and to form metal protein complexes. In mammals, MT are presentedby four isoforms (MT 1, MT 2, MT 3 and MT 4), which are encoded by the family of metallotioneingenes. The purpose of the present study was to determine the p hylogenetic affinity of different ratRattus norvegicus MT genes with other mammalian spe cies, as well as to determine the degree ofsimilarity between genes of MT isoforms within s pecies Rattus norvegicus. Therefore nucleotidesequences of MT genes were aligned, the percentage of identity and the number of gaps wascounted and a phylogenetic analysis was carrie d out. For genes of all Rattus norvegicus MTisoforms, significant similarity with other ma mmalian species Mus musculus Homo s ap iens Susscrofa was shown. The coding sequences ( of genes of Rattus norvegicus MT 1, MT 2A, MT 3and MT 4 isoforms have a significant similarity among themselves. The MT genes are hig hlyconservative, both at the interspecific and intraspecific levels, which is probably associated withtheir biological functions.

Use of Slaughterhouse Sludge in the Bioremediation of an Oxyfluorfen-Polluted Soil

The use of organic matter is a highly accepted environmental practice among scientists for the bioremediation of polluted soils. In this manuscript we study under laboratory conditions the bioremediation capacity of a new biostimulant obtained from slaughterhouse sludge in a soil polluted by the oxyfluorfen at a rate of 4 l ha−1 (manufacturer’s rate recommended) over a 90-day period. We determined its effects on dehydrogenase, urease, β-glucosidase and phosphatase activities, the soil microbial community structure and the evolution of the herbicide in soil. Possibly due to the high content of low molecular weight proteins in the biostimulant, the enzymatic activities were stimulated mainly at the beginning of the experiment. Soil biological parameters were inhibited in oxyfluorfen-polluted soil. At the end of the experiment and compared with the control soil, dehydrogenase, urease, β-glucosidase, and phosphatase activities significantly decreased by 47.8%, 50.5%, 36.4%, and 45.5% in the oxyfluorfen-polluted soil. At 5 days into the experiment, the use of the biostimulant in oxyfluorfen-polluted soils decreased soil enzymatic activities and microbial community inhibition. At the end of the incubation period the oxyfluorfen concentration had decreased by 60% in the polluted soil and amended with biostimulants. These results suggested that the use of this biostimulant with higher amounts of low molecular weight proteins and peptides had a positive effect on the remediating oxyfluorfen-polluted soils. Therefore, this study provides the use of a new biostimulant obtained from slaughterhouse sludge by enzymatic hydrolysis processes used in the bioremediation of a soil polluted by the oxyfluorfen herbicide.

Does Only Egg Sac Protect Spider Embryos From Pathogens? Detection Of Antibacterial Proteins In Embryos of Theridiidae And Lycosidae Representatives

Cocoons covering spider embryos may constitute a physical barrier, protecting eggs from microbial infections. The aim of the study was to find out if the embryos have their own immune potential. We test the effect of cocoon deprivation on the level of antimicrobial proteins (AMPs) produced by spider embryos of Parasteatoda tepidariorum and Pardosa sp. Eggs in the age from 24 to 168 hours were divided in two experimental groups: C (closed, in untouched cocoon) and O (open, embryos isolated from the egg sac). Results indicate that the tested spiders embryos produce lysozyme, defensins and potentially other low-molecular-weight proteins with antimicrobial activity. Level of AMPs increased with the age of spider embryos. Lysozyme in both species was produced at a higher level than defensins. Deprivation of cocoon results in increased production of lysozyme only in Pardosa sp., which may be related to the specific type of parental care of lycosids.

Metallothioneins in Inflammatory Bowel Diseases: Importance in Pathogenesis and Potential Therapy Target

Immunological disorders, increased oxidative stress, and damage to the epithelial barrier play an important role in the pathogenesis of inflammatory bowel diseases (IBDs). In the treatment of patients with Crohn’s disease (CD) and ulcerative colitis (UC), it is increasingly common to use biological drugs that selectively affect individual components of the inflammatory cascade. However, administering the medicines currently available does not always result in obtaining and maintaining remission, and it may also lead to the development of resistance to a given agent over time. Metallothioneins (MTs) belong to the group of low molecular weight proteins, which, among others, regulate the inflammation and homeostasis of heavy metals as well as participating in the regulation of the intensity of oxidative stress. The results of the studies conducted so far do not clearly indicate the role of MTs in the process of inflammation in patients with IBD. However, there are reports that suggest the possibility of using MTs as a potential target in the treatment of this group of patients.