Minimizing the Influence of Fluorescent Tags on IgG Partition in PEG–Salt Aqueous Two‐Phase Systems for Rapid Screening Applications

Aqueous two‐phase extraction (ATPE) has been showing significant potential in the biopharmaceutical industry, allowing the selective separation of high‐value proteins directly from unclarified cell culture supernatants. In this context, effective high‐throughput screening tools are critical to perform a rapid empirical optimization of operating conditions. In particular, microfluidic ATPE screening devices, coupled with fluorescence microscopy to continuously monitor the partition of fluorophore‐labeled proteins, have been recently demonstrated to provide short diffusion distances and rapid partition, using minimal reagent volumes. Nevertheless, the currently overlooked influence of the labeling procedure on partition must be carefully evaluated to validate the extrapolation of results to the unlabeled molecule. Here, three fluorophores with different global charge and reactivity selected to label immunoglobulin G (IgG) at degrees of labeling (DoL) ranging from 0.5 to 7.6. Labeling with BODIPY FL maleimide (DoL = 0.5), combined with tris(2‐carboxyethyl) phosphine (TCEP) to generate free thiol groups, is the most promising strategy to minimize the influence of the fluorophore on partition. In particular, the partition coefficient (K_p) measured in polyethylene glycol (PEG) 3350–phosphate systems with and without the addition of NaCl using microtubes (batch) or microfluidic devices (continuous) is comparable to those quantified for the native protein.     

Extracellular lipid droplets promote hemozoin crystallization in the gut of the blood fluke Schistosoma mansoni

Hemozoin (Hz) is a heme crystal produced upon hemoglobin digestion as the main mechanism of heme disposal in several hematophagous organisms. Here, we show that, in the helminth Schistosoma mansoni, Hz formation occurs in extracellular lipid droplets (LDs). Transmission electron microscopy of adult worms revealed the presence of numerous electron-lucent round structures similar to LDs in gut lumen, where multicrystalline Hz assemblies were found associated to their surfaces. Female regurgitates promoted Hz formation in vitro in reactions partially inhibited by boiling. Fractionation of regurgitates showed that Hz crystallization activity was essentially concentrated on lower density fractions, which have small amounts of pre-formed Hz crystals, suggesting that hydrophilic-hydrophobic interfaces, and not Hz itself, play a key catalytic role in Hz formation in S. mansoni. Thus, these data demonstrate that LDs present in the gut lumen of S. mansoni support Hz formation possibly by allowing association of heme to the lipid-water interface of these structures.     

A simple chromosomal marker can reliably distinguishes <Emphasis Type="Italic">Poncirus</Emphasis> from <Emphasis Type="Italic">Citrus</Emphasis> species

Several chromosome types have been recognized in Citrus and related genera by chromomycin A₃ (CMA) banding patterns and fluorescent in situ hybridization (FISH). They can be used to characterize cultivars and species or as markers in hybridization and backcrossing experiments. In the present work, characterization of six cultivars of P. trifoliata (“Barnes”, “Fawcett”, “Flying Dragon”, “Pomeroy”, “Rubidoux”, “USDA”) and one P. trifoliata × C. limonia hybrid was performed by sequential analyses of CMA banding and FISH using 5S and 45S rDNA as probes. All six cultivars showed a similar CMA⁺ banding pattern with the karyotype formula 4B + 8D + 6F. The capital letters indicate chromosomal types: B, a chromosome with one telomeric and one proximal band; D, with only one telomeric band; F, without bands. In situ hybridization labeling was also similar among cultivars. Three chromosome pairs displayed a closely linked set of 5S and 45S rDNA sites, two of them co-located with the proximal band of the B type chromosomes (B/5S-45S) and the third one co-located with the terminal band of a D pair (D/5S-45S). The B/5S-45S chromosome has never been found in any citrus accessions investigated so far. Therefore, this B chromosome can be used as a marker to recognize the intergeneric Poncirus × Citrus hybrids. The intergeneric hybrid analyzed here displayed the karyotype formula 4B + 8D + 6F, with two chromosome types B/5S-45S and two D/5S-45S. The karyotype formula and the presence of two B/5S-45S chromosomes clearly indicate that the plant investigated is a symmetric hybrid. It also demonstrates the suitability of karyotype analyses to differentiate zygotic embryos or somatic cell fusions involving trifoliate orange germplasm. During the submission of this paper, we analyzed 25 other citrus cultivars with the same methodology and we found that the chromosome marker reported here can indeed distinguish Poncirus trifoliata from grapefruits, pummelos, and one variegated access of Citrus, besides the previously reported access of limes, limons, citrons, and sweet-oranges. However, among 14 mandarin cultivars, two of them displayed a single B/5S-45S chromosome, whereas in Citrus hystrix D.C., a far related species belonging to the Papeda subgenus, this chromosome type was found in homozygosis. Since these two mandarin cultivars are probably of hybrid origin, we assume that for almost all commercial cultivars and species of the subgenus Citrus this B type chromosome is a useful genetic marker.     

The lactate dehydrogenases encoded by the ldh and ldhB genes in Lactococcus lactis exhibit distinct regulation and catalytic properties - comparative modeling to probe the molecular basis

Lactococcus lactis FI9078, a construct carrying a disruption of the ldh gene, converted approximately 90% of glucose into lactic acid, like the parental strain MG1363. This unexpected lactate dehydrogenase activity was purified, and ldhB was identified as the gene encoding this protein. The activation of ldhB was explained by the insertion of an IS905-like element that created a hybrid promoter in the intergenic region upstream of ldhB. The biochemical and kinetic properties of this alternative lactate dehydrogenase (LDHB) were compared to those of the ldh-encoded enzyme (LDH), purified from the parental strain. In contrast to LDH, the affinity of LDHB for NADH and the activation constant for fructose 1,6-bisphosphate were strongly dependent on pH. The activation constant increased 700-fold, whereas the K(m) for NADH increased more than 10-fold, in the pH range 5.5-7.2. The two enzymes also exhibited different pH profiles for maximal activity. Moreover, inorganic phosphate acted as a strong activator of LDHB. The impact of replacing LDH by LDHB on the physiology of L. lactis was assessed by monitoring the evolution of the pools of glycolytic intermediates and cofactors during the metabolism of glucose by in vivo NMR. Structural analysis by comparative modeling of the two proteins showed that LDH has a slightly larger negative charge than LDHB and a greater concentration of positive charges at the interface between monomers. The calculated pH titration curves of the catalytic histidine residues explain why LDH maintains its activity at low pH as compared to LDHB, the histidines in LDH showing larger pH titration ranges.     

Simple and rapid method determination for metformin in human plasma using high performance liquid chromatography tandem mass spectrometry: application to pharmacokinetic studies

A rapid and simple method for quantitation of metformin (MET) in human plasma by HPLC-MS/MS was developed and validated. The sample preparation consists of plasma deproteinization using acetonitrile. The mobile phase consisted of water-acetonitrile and formic acid (55/45/0.048, v/v/%) and the run time was 3 min. A pursuit C(18) (100 mm x 2.0 mm i.d., 3 microm) column connected to a guard column MS-pursuit (0.20 mm x 0.20 mm i.d., 5 microm) was used. The range of the calibration curve was from 20 to 5000 ng/mL, the limit of quantitation being 20 ng/mL. The detection was performed on a mass spectrometer (ESI+), using metoprolol as internal standard. The calibration curves have r(2) values of 0.995 (CV=0.24%, n=10). The accuracy and precision were between 90.74 and 106.7% and coefficients of variations (CV) of 1.10 and 4.35%, respectively. The method was applied to determine the pharmacokinetic parameters: C(max) (1667.25 ng/mL) and T(max) (3.89 h).     

Analysis of the Paracoccidioides brasiliensis triosephosphate isomerase suggests the potential for adhesin function

Paracoccidioides brasiliensis is an important fungal pathogen. The disease it causes, paracoccidioidomycosis (PCM), ranges from localized pulmonary infection to systemic processes that endanger the life of the patient. Paracoccidioides brasiliensis adhesion to host tissues contributes to its virulence, but we know relatively little about molecules and the molecular mechanisms governing fungal adhesion to mammalian cells. Triosephosphate isomerase (TPI: EC 5.3.1.1) of P. brasiliensis (PbTPI) is a fungal antigen characterized by microsequencing of peptides. The protein, which is predominantly expressed in the yeast parasitic phase, localizes at the cell wall and in the cytoplasmic compartment. TPI and the respective polyclonal antibody produced against this protein inhibited the interaction of P. brasiliensis to in vitro cultured epithelial cells. TPI binds preferentially to laminin, as determined by peptide inhibition assays. Collectively, these results suggest that TPI is required for interactions between P. brasiliensis and extracellular matrix molecules such as laminin and that this interaction may play an important role in the fungal adherence and invasion of host cells.     

GSK3β/eEF2K信号通路对小鼠肺纤维化过程的影响*

目的: 探讨糖原合成酶激酶-3(GSK3β)/真核延伸因子激酶2(eEF2K)信号通路对肺纤维化进程的影响,为临床治疗肺纤维化寻找新的思路。方法: 采用一次性气管注射法构建C57BL/6雄性小鼠博莱霉素肺纤维化模型,造模14 d后将动物分成模型组、阴性抑制组与抑制组(n=5),另设空白组不作处理。抑制组使用腹腔注射TDZD-8(4 mg/kg),阴性抑制组腹腔注射二甲基亚砜(DMSO)溶液,28 d后处死采集指标。采用苏木精-伊红染色法检测小鼠肺脏病变情况;试剂盒水解法检测肺组织中羟脯氨酸(Hyp)的含量;采用Western blot法检测肺脏中GSK3β、磷酸化GSK3β(p-GSK3β)、eEF2K、p-eEF2K(Ser70)、p-eEF2K(Ser392)、p-eEF2K(Ser470)、基质金属蛋白酶-2前体蛋白(pro-MMP-2)、基质金属蛋白酶-2(MMP-2)蛋白表达水平,使用免疫组织化学法检测肺脏中MMP-2、胶原蛋白I(Col I)、胶原蛋白Ⅲ(Col Ⅲ)、α-平滑肌蛋白(α-SMA)的表达。结果: 与空白组相比,模型组中GSK3β、p-GSK3β、p-eEF2K(Ser70)、p-eEF2K(Ser392)、p-eEF2K(Ser470)、pro-MMP-2、MMP-2、Col I、Col Ⅲ、α-SMA蛋白表达水平升高,eEF2K蛋白表达水平降低(P＜0.05);与模型组相比,抑制组GSK3β、p-GSK3β、p-eEF2K(Ser70)、p-eEF2K(Ser392)、p-eEF2K(Ser470)、pro-MMP-2、MMP-2、Col I、Col Ⅲ、α-SMA蛋白表达降低,eEF2K蛋白表达升高(P＜ 0.05)。结论: GSK3β能通过Ser70、Ser392、Ser470这3个位点磷酸化激活eEF2K,增加纤维化指标含量,促进肺纤维化形成,加重肺组织病变。     

Bait traps remain attractive to euglossine bees even after two weeks: a report from Brazilian Atlantic forest

Bait traps are effective and commonly used method in the studies of orchid bees. Important questions in the context of this method, including those related to bait dispersion, how long baits remain attractive, the distance from which males are supposed to be attracted to lures and so on, are still open subjects. Data on the attractiveness of bait traps that have remained in the field during two weeks in a large Atlantic forest preserve are presented. Four main results emerge from the data: (i) the abundance of specimens collected per day decreased in all the attractants as the traps were left on the field; (ii) despite this decrease, the absolute number of individuals collected after eight and fifteen days is remarkably, mostly in eugenol and vanillin baits; (iii) the vast majority of species, 22 of 25, was already collected on the first sample day; (iv) a consistent variation in the relative abundance of individuals collected in each scent as collections were made. We urge that bait traps should not be left in the field beyond what is strictly necessary since there is a real possibility of collecting a significant number of individuals as these traps remain available.     

The mitochondrial Nod-like receptor NLRX1 modifies apoptosis through SARM1

Molecular and Cellular Biochemistry - Acrolein is a α-β-unsaturated aldehyde and is toxic to human upon its exposure from the environment. Sources of exposure to acrolein can be from...