A loop of coagulation factor VIIa influencing macromolecular substrate specificity

Coagulation factor VIIa (FVIIa) belongs to a family of proteases being part of the stepwise, self-amplifying blood coagulation cascade. To investigate the impact of the mutation Met(298{156})Lys in FVIIa, we replaced the Gly(283{140})-Met(298{156}) loop with the corresponding loop of factor Xa. The resulting variant exhibited increased intrinsic activity, concurrent with maturation of the active site, a less accessible N-terminus, and, interestingly, an altered macromolecular substrate specificity reflected in an increased ability to cleave factor IX (FIX) and a decreased rate of FX activation compared to that of wild-type FVIIa. In complex with tissue factor, activation of FIX, but not of FX, returned to normal. Deconvolution of the loop graft in order to identify important side chain substitutions resulted in the mutant Val(158{21})Asp/Leu(287{144})Thr/Ala(294{152})Ser/Glu(296{154}) Ile/Met(298{156})Lys-FVIIa with almost the same activity and specificity profile. We conclude that a lysine residue in position 298{156} of FVIIa requires a hydrophilic environment to be fully accommodated. This position appears critical for substrate specificity among the proteases of the blood coagulation cascade due to its prominent position in the macromolecular exosite and possibly via its interaction with the corresponding position in the substrate (i.e. FIX or FX).     

Allosteric Inhibition as a New Mode of Action for BAY 1213790, a Neutralizing Antibody Targeting the Activated Form of Coagulation Factor XI

Factor XI (FXI), the zymogen of activated FXI (FXIa), is an attractive target for novel anticoagulants because FXI inhibition offers the potential to reduce thrombosis risk while minimizing the risk of bleeding. BAY 1213790, a novel anti-FXIa antibody, was generated using phage display technology. Crystal structure analysis of the FXIa–BAY 1213790 complex demonstrated that the tyrosine-rich complementarity-determining region 3 loop of the heavy chain of BAY 1213790 penetrated deepest into the FXIa binding epitope, forming a network of favorable interactions including a direct hydrogen bond from Tyr102 to the Gln451 sidechain (2.9 Å). The newly discovered binding epitope caused a structural rearrangement of the FXIa active site, revealing a novel allosteric mechanism of FXIa inhibition by BAY 1213790. BAY 1213790 specifically inhibited FXIa with a binding affinity of 2.4 nM, and in human plasma, prolonged activated partial thromboplastin time and inhibited thrombin generation in a concentration-dependent manner.     

Carbon cycle of larch plantation based on CO2FIX model

Aims
Plantations play important roles in modifying regional carbon budget and maintaining regional carbon balance. In this study, we assessed larch plantation (Larix gmelinii var. principis-rupprechtii) carbon dynamics in Weichang County from a perspective of the forest biomass-soil-wood-products chain. Our objectives were to elucidate the carbon sink capacity of larch plantation and the influences of biomass, soil and wood product pools on carbon balance.
Methods
CO2FIX model was used to evaluate the carbon storage and flow of larch plantation over a time span of 120 years. Input data for model were derived from practical investigations and published papers. We validated the simulated results and found that this model was suitable in the region and the simulated results were reliable.
Important findings
(1) Soil was the largest carbon pool for larch plantation and the wood product pool had the smallest carbon storage. Meanwhile, carbon storage in wood products gradually increased with time. (2) In a rotation of 50 years from secondary poplar-birch forest to larch plantation, 250 t C·hm^-2 was sequestrated by the larch plantation. 70% of the carbon was transferred into soil in the form of litter and logging slash and the other 30% was transferred into wood products. (3) Larch plantation was a carbon sink during most of its growing period and turned to temporary carbon source when it was harvested. Larch plantation could sequestrate about 0.3 t C·hm^-2·a^-1 in the long term. Our results indicated the importance of wood product carbon pool in carbon dynamics of plantation, which facilitated our understanding in the carbon dynamics and capacity of plantation.     

NUCEL (Cell and Molecular Therapy Center): A Multidisciplinary Center for Translational Research in Brazil

Social and economical development is closely associated with technological innovation and a well-developed biotechnological industry. In the last few years, Brazil’s scientific production has been steadily increasing; however, the number of patents is lagging behind, with technological and translational research requiring governmental incentive and reinforcement. The Cell and Molecular Therapy Center (NUCEL) was created to develop activities in the translational research field, addressing concrete problems found in biomedical and veterinary areas and actively searching for solutions by employing a genetic engineering approach to generate cell lines over-expressing recombinant proteins to be transferred to local biotech companies, aiming at furthering the development of a national competence for local production of biopharmaceuticals of widespread use and of life-saving importance. To this end, mammalian cell engineering technologies were used to generate cell lines over-expressing several different recombinant proteins of biomedical and biotechnological interest, namely, recombinant human Amylin/IAPP for diabetes treatment, human FVIII and FIX clotting factors for hemophilia, human and bovine FSH for fertility and reproduction, and human bone repair proteins (BMPs). Expression of some of these proteins is also being sought with the baculovirus/insect cell system (BEVS) which, in many cases, is able to deliver high-yield production of recombinant proteins with biological activity comparable to that of mammalian systems, but in a much more cost-effective manner. Transfer of some of these recombinant products to local Biotech companies has been pursued by taking advantage of the São Paulo State Foundation (FAPESP) and Federal Government (FINEP, CNPq) incentives for joint Research Development and Innovation partnership projects.     

VIS2FIX: a high-speed fixation method for immuno-electron microscopy

Immuno-transmission electron microscopy (TEM) is the technique of choice for high-resolution localization of proteins in fixed specimen. Here we introduce 2 novel methods for the fixation of sections from cryo-immobilized samples that result in excellent ultrastructural preservation. These high-speed fixation techniques, both called VIS2FIX, allow for a reduction in sample preparation time from at least 1 week to only 8 h. The methods were validated in immuno-TEM experiments on THP-1 monocytes, human umbilical vein endothelial cells (HUVECs) and Madin-Darby canine kidney (MDCK-II) cells. The fixation and retention of neutral lipids is demonstrated, offering unique prospects for the application of immuno-TEM in the lipidomics field. Furthermore, the VIS2FIX methods were successfully employed in correlative fluorescence and electron microscopy.     

Development of hybrid viral vectors for gene therapy

Adenoviral, retroviral/lentiviral, adeno-associated viral, and herpesviral vectors are the major viral vectors used in gene therapy. Compared with non-viral methods, viruses are highly-evolved, natural delivery agents for genetic materials. Despite their remarkable transduction efficiency, both clinical trials and laboratory experiments have suggested that viral vectors have inherent shortcomings for gene therapy, including limited loading capacity, immunogenicity, genotoxicity, and failure to support long-term adequate transgenic expression. One of the key issues in viral gene therapy is the state of the delivered genetic material in transduced cells. To address genotoxicity and improve the therapeutic transgene expression profile, construction of hybrid vectors have recently been developed. By adding new abilities or replacing certain undesirable elements, novel hybrid viral vectors are expected to outperform their conventional counterparts with improved safety and enhanced therapeutic efficacy. This review provides a comprehensive summary of current achievements in hybrid viral vector development and their impact on the field of gene therapy.     

Tick salivary secretion as a source of antihemostatics

Ticks are mostly obligatory blood feeding ectoparasites that have an impact on human and animal health. In addition to direct damage due to feeding, some tick species serve as the vectors for the causative agents of several diseases, such as the spirochetes of the genus Borrelia causing Lyme disease, the virus of tick-borne encephalitis, various Rickettsial pathogens or even protozoan parasites like Babesia spp. Hard ticks are unique among bloodfeeders because of their prolonged feeding period that may last up to two weeks. During such a long period of blood uptake, the host develops a wide range of mechanisms to prevent blood loss. The arthropod ectoparasite, in turn, secretes saliva in the sites of bite that assists blood feeding. Indeed, tick saliva represents a rich source of proteins with potent pharmacologic action that target different mechanisms of coagulation, platelet aggregation and vasoconstriction. Tick adaptation to their vertebrate hosts led to the inclusion of a powerful protein armamentarium in their salivary secretion that has been investigated by high-throughput methods. The resulting knowledge can be exploited for the isolation of novel antihemostatic agents. Here we review the tick salivary antihemostatics and their characterized functions at the molecular and cellular levels.     

Carbon cycle of larch plantation based on CO2FIX model北大核心CSCD

Aims Plantations play important roles in modifying regional carbon budget and maintaining regional carbon balance. In this study, we assessed larch plantation (Larix gmelinii var. principis-rupprechtii) carbon dynamics in Weichang County from a perspective of the forest biomass-soil-wood-products chain. Our objectives were to elucidate the carbon sink capacity of larch plantation and the influences of biomass, soil and wood product pools on carbon balance. Methods CO2FIX model was used to evaluate the carbon storage and flow of larch plantation over a time span of 120 years. Input data for model were derived from practical investigations and published papers. We validated the simulated results and found that this model was suitable in the region and the simulated results were reliable. Important findings (1) Soil was the largest carbon pool for larch plantation and the wood product pool had the smallest carbon storage. Meanwhile, carbon storage in wood products gradually increased with time. (2) In a rotation of 50 years from secondary poplar-birch forest to larch plantation, 250 t C•hm2 was sequestrated by the larch plantation. 70% of the carbon was transferred into soil in the form of litter and logging slash and the other 30% was transferred into wood products. (3) Larch plantation was a carbon sink during most of its growing period and turned to temporary carbon source when it was harvested. Larch plantation could sequestrate about 0.3 t C•hm2•a1 in the long term. Our results indicated the importance of wood product carbon pool in carbon dynamics of plantation, which facilitated our understanding in the carbon dynamics and capacity of plantation.     

基于CO2FIX模型的长白落叶松人工林碳汇和木材生产模拟

本研究以帽儿山地区长白落叶松人工林为对象,基于样地调查和文献数据,利用CO₂FIX模型定量模拟轮伐期(30、40、50、60年)、立地指数(12、16、20 m)和初植密度(2500、3333、4444 株·hm^-2)对长白落叶松人工林碳平衡过程的影响,并构建林分尺度下生物量碳库、土壤碳库和林产品碳库之间的碳流通过程。结果表明: CO₂FIX模型对帽儿山地区长白落叶松人工林生物量和蓄积量的生长过程模拟结果具有较高的可靠性,模拟值和实测值平均相对误差分别为6.4%和3.7%。在初植密度3333 株·hm^-2、立地指数16 m、轮伐期40年的基准条件下,长白落叶松人工林总碳储量及各碳库碳储量均随轮伐期呈周期性变化。林分总碳储量和蓄积量均随轮伐期的延长、立地指数的提升和初植密度的增加而增加。当轮伐期分别延长10年和20年时,林分碳储量分别增加12.2%和31.2%,林分蓄积增加36.7%和67.8%;而当轮伐期缩短10年时,林分碳储量和蓄积量则分别降低20.9%和40.4%。与初植密度2500 株·hm^-2相比,初植密度为3333和4444 株·hm^-2时,林分碳储量增长率分别为27.8%和50.9%,蓄积量增长率分别为27.4%和49.1%。当立地指数在12～20 m范围时,每提高4 m,林分碳储量增长36.0%、40.3%,蓄积量增长39.3%、44.2%。在一个轮伐期内,每公顷长白落叶松人工林可固定约271.57 t C;当轮伐期结束时,约有27.47和56.75 t C流转到土壤和木材产品碳库中。因此,当立地条件较好时,采用较高初植密度(4444 株·hm^-2)和较长轮伐期(60年)的管理模式更有利于长白落叶松人工林碳汇和木材效益的最大化。     

基于CO2FIX模型的华北落叶松人工林碳循环过程

为了探明华北落叶松(Larix gmelinii var. principis-rupprechtii)人工林的碳循环过程, 该研究以河北围场地区的华北落叶松人工林为例, 基于CO2FIX模型, 以在当地的实际调查数据、文献数据作为输入数据, 从生物量、土壤和木质林产品碳库3个方面探讨了华北落叶松人工林的碳循环过程和碳汇能力。结果表明: 华北落叶松人工林土壤碳库最大, 生物量碳库次之, 林产品碳库最小, 但是林产品碳库随时间呈逐渐增加的趋势; 在一个轮伐期内(50年), 每公顷华北落叶松人工林约固定了250 t碳, 其中约70%通过凋落物和采伐剩余物的方式进入土壤碳库, 约30%进入木质林产品碳库; 华北落叶松人工林在生长的大部分时间是一个碳的吸收汇, 而在森林采伐时成为暂时的排放源, 从长时间尺度上看, 每公顷华北落叶松人工林每年大约固定0.3 t左右的碳。该研究结果表明了木质林产品碳库在人工林碳循环中的重要作用, 这将有助于更加全面地认识人工林的碳循环过程和碳汇能力。