An Integrative Structural Biology Analysis of Von Willebrand Factor Binding and Processing by ADAMTS-13 in Solution

Von Willebrand Factor (vWF), a 300-kDa plasma protein key to homeostasis, is cleaved at a single site by multi-domain metallopeptidase ADAMTS-13. vWF is the only known substrate of this peptidase, which circulates in a latent form and becomes allosterically activated by substrate binding. Herein, we characterised the complex formed by a competent peptidase construct (AD13-MDTCS) comprising metallopeptidase (M), disintegrin-like (D), thrombospondin (T), cysteine-rich (C), and spacer (S) domains, with a 73-residue functionally relevant vWF-peptide, using nine complementary techniques. Pull-down assays, gel electrophoresis, and surface plasmon resonance revealed tight binding with sub-micromolar affinity. Cross-linking mass spectrometry with four reagents showed that, within the peptidase, domain D approaches M, C, and S. S is positioned close to M and C, and the peptide contacts all domains. Hydrogen/deuterium exchange mass spectrometry revealed strong and weak protection for C/D and M/S, respectively. Structural analysis by multi-angle laser light scattering and small-angle X-ray scattering in solution revealed that the enzyme adopted highly flexible unbound, latent structures and peptide-bound, active structures that differed from the AD13-MDTCS crystal structure. Moreover, the peptide behaved like a self-avoiding random chain. We integrated the results with computational approaches, derived an ensemble of structures that collectively satisfied all experimental restraints, and discussed the functional implications. The interaction conforms to a ‘fuzzy complex’ that follows a ‘dynamic zipper’ mechanism involving numerous reversible, weak but additive interactions that result in strong binding and cleavage. Our findings contribute to illuminating the biochemistry of the vWF:ADAMTS-13 axis.     

Ultrasound shear wave elastography measurement of the deep posterior cervical muscles: Reliability and ability to differentiate between muscle contraction states

The deep posterior cervical muscles (DPCM), specifically the semispinalis cervicis and cervical multifidus, are often impaired in patients with neck disorders and have been assessed by several imaging techniques. Prior ultrasound shear wave elastography (SWE) imaging and reliability assessments of the DPCM were performed utilizing similar positioning as assessments for the more superficial cervical extensors. Our objectives were to describe an SWE imaging technique for the DPCM, establish intra-rater reliability of DPCM SWE, and compare DPCM shear modulus during rest and submaximal contraction in both prone and seated positions in individuals without spinal pain. In sixteen participants, the DPCM was located using B‐mode ultrasound, then muscle shear modulus was assessed via SWE at both rest and with contraction against a 2‐kg resistance applied at the C2 spinous process. Within‐day intra‐rater reliability was moderate to good (ICC = 0.70–0.88). The DPCM were stiffer during contraction than at rest in the prone position (p = 0.002), and at rest in sitting versus at rest in prone (p = 0.003). Further research is needed to assess DPCM-specific SWE in symptomatic individuals and compare DPCM shear modulus to electromyography across contraction intensities.     

Enhancement of Lanthanum (III) on Sodium Currents in Acutely Isolated Hippocampal CA1 Neurons of Rat

The effects of lanthanum (III) (La³⁺) on voltage-gated sodium channel currents (I _Na) in freshly dissociated rat hippocampal CA1 neurons were studied using the whole-cell patch clamp techniques. La³⁺ reversibly enhanced I _Na in a concentration- and voltage-dependent manner. The 50% enhancement concentration (EC₅₀) of La³⁺ on I _Na was 9.93 μM. In addition, 10 μM La³⁺ shifted the steady state activation curve of I _Na towards positive potential and the steady state inactivation curve towards negative potential without changing the slope factor. These results indicated that La³⁺ could increase the amplitudes of I _Na and change the activation and inactivation courses of I _Na even in very low concentration.     

Generating transgenic mice from bacterial artificial chromosomes: transgenesis efficiency, integration and expression outcomes

Transgenic mice are widely used in biomedical research to study gene expression, developmental biology, and gene therapy models. Bacterial artificial chromosome (BAC) transgenes direct gene expression at physiological levels with the same developmental timing and expression patterns as endogenous genes in transgenic animal models. We generated 707 transgenic founders from 86 BAC transgenes purified by three different methods. Transgenesis efficiency was the same for all BAC DNA purification methods. Polyamine microinjection buffer was essential for successful integration of intact BAC transgenes. There was no correlation between BAC size and transgenic rate, birth rate, or transgenic efficiency. A narrow DNA concentration range generated the best transgenic efficiency. High DNA concentrations reduced birth rates while very low concentrations resulted in higher birth rates and lower transgenic efficiency. Founders with complete BAC integrations were observed in all 47 BACs for which multiple markers were tested. Additional founders with BAC fragment integrations were observed for 65% of these BACs. Expression data was available for 79 BAC transgenes and expression was observed in transgenic founders from 63 BACs (80%). Consistent and reproducible success in BAC transgenesis required the combination of careful DNA purification, the use of polyamine buffer, and sensitive genotyping assays.     

Stratégies techniques dans le Paléolithique Moyen du sud-est des Pyrénées

In this article, the lithic reduction systems from Middle Paleolithic levels at Roca dels Bous and Tragó are presented. These two sites are located in the South-eastern Pyrenees in Catalunya (Spain) and yield Mousterian levels which are attributed to MIS 3 and MIS 5. At the two studied sites, there is coexistence between expedient knapping systems and more complex techniques such as the Levallois method. Furthermore, cores are heavily exhausted, showing a pattern that cannot be explained by the absence or scarcity of raw material. This technical pattern can be traced across several Mousterian assemblages in the South-eastern Pyrenees, suggesting technocognitive continuity in the Middle Palaeolithic during the Upper Pleistocene, in which changes in lithic reduction patterns are not evident. In this paper, the implications of such observations are contextualized within the general discussion on the behaviour of the South-eastern Pyrenees Neanderthals.     

Collecting isolated microvertebrate fossils

A review is presented of the techniques currently used in the collection and separation of isolated teeth and bones of fossil vertebrates. These involve the collection and disaggregation of the sediment, its sieving, concentration and sorting of the residue, and curation of the fossils obtained.     

ISOLATION AND REGENERATION OF HAPLOID PROTOPLASTS FROM BANGIA ATROPURPUREA (RHODOPHYTA) WITH MARINE BACTERIAL ENZYMES1

Three kinds of enzymes, agarase, β-1,4-mannanase, and β-1,3-xylanase, required for isolation of protoplasts from the red alga Bangia atropurpurea (Roth) C. Ag. were prepared from bacterial culture fluids of Vibrio sp. PO-303, Vibrio sp. MA-138, and Alcaligenes sp. XY-234, respectively, isolated from the sea environment. The optimal pH of all enzymes was around 7.5. Suitable conditions for protoplast isolation from B. atropurpurea were examined. The pretreatment of the fronds with pa-pain solution (20 mM Mes buffer, pH 7.5, containing 2% papain and 0.5 M mannitol) contributed to successful protoplast isolation. When razor-cut fragments of the fronds (about 200 mg in fresh weight) immersed in 20 mM Mes buffer, 7.5, containing 0.5 M mannitol and one unit each of agarase, β-1,4-mannanase, and β-1,3-xylanase were incubated at 22°C for 90 min with gentle agitation, 5.7 × 10⁶ protoplasts were released from them. Many protoplasts regenerated into fronds of regular or irregular shape.     

金针虫调查方法及评价

金针虫是一类重要的地下害虫,其种群数量调查是植物保护工作中的难点之一。文章概述金针虫种群数量调查的3种主要方法:土方抽样、诱饵诱捕及成虫性信息素诱捕。就其调查原理、特点及研究现状进行了介绍,并对各方法的不足及应用前景做了简要分析。     

A new paradigm for the action of reactive oxygen species in the photoinhibition of photosystem II

Inhibition of the activity of photosystem II (PSII) under strong light is referred to as photoinhibition. This phenomenon is due to the imbalance between the rate of photodamage to PSII and the rate of the repair of damaged PSII. Photodamage is initiated by the direct effects of light on the oxygen-evolving complex and, thus, photodamage to PSII is unavoidable. Studies of the effects of oxidative stress on photodamage and subsequent repair have revealed that reactive oxygen species (ROS) act primarily by inhibiting the repair of photodamaged PSII and not by damaging PSII directly. Thus, strong light has two distinct effects on PSII; it damages PSII directly and it inhibits the repair of PSII via production of ROS. Investigations of the ROS-induced inhibition of repair have demonstrated that ROS suppress the synthesis de novo of proteins and, in particular, of the D1 protein, that are required for the repair of PSII. Moreover, a primary target for inhibition by ROS appears to be the elongation step of translation. Inhibition of the repair of PSII by ROS is accelerated by the deceleration of the Calvin cycle that occurs when the availability of CO₂ is limited. In this review, we present a new paradigm for the action of ROS in photoinhibition.