Machine Learning-Assisted High-Throughput Identification and Quantification of Protein Biomarkers with Printed Heterochains.

Advanced in vitro diagnosis technologies are highly desirable in early detection, prognosis, and progression monitoring of diseases. Here, we engineer a multiplex protein biosensing strategy based on the tunable liquid confinement self-assembly of multi-material heterochains, which show improved sensitivity, throughput, and accuracy compared to standard ELISA kits. By controlling the material combination and the number of ligand nanoparticles (NPs), we observe robust near-field enhancement as well as both strong electromagnetic resonance in polymer-semiconductor heterochains. In particular, their optical signals show a linear response to the coordination number of the semiconductor NPs in a wide range. Accordingly, a visible nanophotonic biosensor is developed by functionalizing antibodies on central polymer chains that can identify target proteins attached to semiconductor NPs. This allows for the specific detection of multiple protein biomarkers from healthy people and pancreatic cancer patients in one step with an ultralow detection limit (1 pg/mL). Furthermore, rapid and high-throughput quantification of protein expression levels in diverse clinical samples such as buffer, urine, and serum is achieved by combining a neural network algorithm, with an average accuracy of 97.3%. This work demonstrates that the heterochain-based biosensor is an exemplary candidate for constructing next-generation diagnostic tools and suitable for many clinical settings.

A dual-reference study design for understanding and improving AAV genome size analysis.

Recombinant adeno-associated virus (rAAV) is the leading platform of gene delivery for its long-lasting gene transformation and low immunogenicity. Characterization of the integrity and purity of the rAAV genome is critical to ensure clinical potency and safety. However, current rAAV genome characterization methods that can provide size assessment are either time-consuming or not easily accessible to general labs. Additionally, there is a lack of right reference standard for analyzing long single-stranded DNA (ssDNA) fragments. Here, we have developed an ssDNA assay on a microfluidic capillary electrophoresis platform using ssDNA reference standard. This assay provides size calling for ssDNA fragment, a detection sensitivity at ∼89 pg/µL (3 × 1010  GC/mL AAV) for 5.1 kb ssDNA fragment, and a turnaround time at ∼100 s per sample with a high throughput sample analyzing capability. Moreover, we have observed that the annealing of AAV ssDNA subsequent to its release from the capsid might introduce an additional double-stranded DNA (dsDNA) peak. This phenomenon is dependent on the sample processing workflow. To avoid the risk of mischaracterization, we recommend the use of dual-reference standards in combination with other orthogonal methods to have a comprehensive understanding of the rAAV genome size and integrity.

Exogenous brassinolide treatment regulates phenolic accumulation in mung bean sprouts through the modulation of sugar and energy metabolism.

BACKGROUND: The effects of exogenous brassinolide (BR) treatment (3.0 µmol L-1 ) on phenolic biosynthesis in mung bean sprouts were investigated. This investigation included the analysis of sugar content, substrates within the phenylpropane pathway, energy substances, enzymatic activity within the phenylpropane pathway, sugar metabolism and energy metabolism. RESULTS: Results showed that BR treatment significantly increased the levels of total phenolics, p-hydroxybenzoic acid, p-coumaric acid, gallic acid, fumalic acid and caffeic acid. This enhancement was accomplished through the elevation of l-phenylalanine levels and the activation of enzymes associated with the phenylpropane pathway in mung bean sprouts, including phenylalanine ammonia-lyase, cinnamate 4-hydroxylase and 4-coumarate CoA ligase. Furthermore, BR treatment induced alterations in sugar metabolism in mung bean sprouts as evidenced by the increased levels of glucose, fructose, sucrose and phosphoenolpyruvate. Moreover, increased activity was observed for enzymes linked to sucrose metabolism and glycolysis in the BR-treated group. Concurrently, BR treatment bolstered the levels of adenosine triphosphate and energy charge in mung bean sprouts, which was attributed to the activation of H+ -adenosine triphosphatase, Ca2+ -adenosine triphosphatase and succinic dehydrogenase. CONCLUSION: These results suggest that BR treatment can accelerate the accumulation of phenolic compounds in mung bean sprouts. This effect is achieved not only through the activation of the phenylpropane pathway, but also through the modulation of sugar and energy metabolism. The modulation provides ample energy and a substrate for the biosynthesis of phenolics. © 2023 Society of Chemical Industry.

Effects of lane-change scenarios on lane-change decision and eye movement.

Improper lane-change manoeuvre can cause traffic safety issues and even lead to serious traffic collisions. Quantifying the decision behaviour and eye movements can provide a deeper understanding of lane-change manoeuvre in vehicle interaction environment. The purpose of this study was to investigate the effect of lane-change scenarios defined by gaps on lane-change decision and eye movements. Twenty-eight participants were recruited to complete a naturalistic driving experiment. Eye movements and lane-change decision duration (LDD) were recorded and analysed. Results suggested that the scanning frequency (SF) and saccade duration (SD) were the sensitive parameters to respond to lane-change scenarios. LDD was significantly affected by the scenario, SF, and SD. The increase in LDD was related to the high difficulty gap and high frequency scanning of multiple regions. These findings evaluated the driver's decision performance in response to different lane-change environments and provided valuable information for measuring the driver's scenario perception ability.Practitioner summary: A naturalistic driving experiment was conducted to evaluate the interaction of lane-change decision, eye movement, and lane changing gap in a lane-change task. The results reveal the sensitive eye movement parameters to lane-change scenario, which provide guidelines for driver's perception ability test and professional driver assessment.

Molecular Recognition-Modulated Hetero-Assembly of Nanostructures for Visualizable and Portable Detection of Circulating miRNAs.

Biomolecular markers, particularly circulating microRNAs (miRNAs) play an important role in diagnosis, monitoring, and therapeutic intervention of cancers. However, existing detection strategies remain intricate, laborious, and far from being developed for point-of-care testing. Here, we report a portable colorimetric sensor that utilizes the hetero-assembly of nanostructures driven by base pairing and recognition for direct detection of miRNAs. Following hybridization, two sizes of nanoparticles modified with single-strand DNA can be robustly assembled into heterostructures with strong optical resonance, exhibiting distinct structure colors. Particularly, the large nanoparticles are first arranged into nanochains to enhance scattering signals of small nanoparticles, which allows for sensitive detection and quantification of miRNAs without the requirement of target extraction, amplification, and fluorescent labels. Furthermore, we demonstrate the high specificity and single-base selectivity of testing different miRNA samples, which shows great potential in the diagnosis, staging, and monitoring of cancers. These heterogeneous assembled nanostructures provide an opportunity to develop simple, fast, and convenient tools for miRNAs detection, which is suitable for many scenarios, especially in low-resource setting.

Toward High Efficient Cu2 ZnSn(Sx ,Se1- x )4 Solar Cells: Break the Limitations of VOC and FF.

Increasing the fill factor (FF) and the open-circuit voltage (VOC ) simultaneously together with non-decreased short-circuit current density (JSC ) are a challenge for highly efficient Cu2 ZnSn(S,Se)4 (CZTSSe) solar cells. Aimed at such target in CZTSSe solar cells, a synergistic strategy to tailor the recombination in the bulk and at the heterojunction interface has been developed, consisting of atomic-layer deposited aluminum oxide (ALD-Al2 O3 ) and (NH4 )2 S treatment. With this strategy, deep-level CuZn defects are converted into shallower VCu defects and improved crystallinity, while the surface of the absorber is optimized by removing Zn- and Sn-related impurities and incorporating S. Consequently, the defects responsible for recombination in the bulk and at the heterojunction interface are effectively passivated, thereby prolonging the minority carrier lifetime and increasing the depletion region width, which promote carrier collection and reduce charge loss. As a consequence, the VOC deficit decreases from 0.607 to 0.547 V, and the average FF increases from 64.2% to 69.7%, especially, JSC does not decrease. Thus, the CZTSSe solar cell with the remarkable efficiency of 13.0% is fabricated. This study highlights the increased FF together with VOC simultaneously to promote the efficiency of CZTSSe solar cells, which could also be applied to other photoelectronic devices.

Rhodocybe subasyae, a new species of Rhodocybe sect. Rufobrunnea (Entolomataceae, Agaricales) from northeast China.

Rhodocybe subasyae is described from northeast China as a new species with both morphological and molecular characteristics, which is morphologically characterized by its tricholomatoid basidiomata, orange white to beige red pileus, adnexed to sinuate lamellae and long clavate branched cheilocystidia within section Rufobrunnea. The phylogenetic tree based on internal transcribed spacers of rDNA (nrITS) sequences using Bayesian methods showed that new taxon was distinguished from other species of Rhodocybe.

Transient Thermal Analysis Model of Damaged Bearing Considering Thermo-Solid Coupling Effect.

As one of the important parameters of bearing operation, temperature is a key metric to diagnose the state of service of a bearing. However, there are still some shortcomings in the study of the temperature variation law for damaged bearings. In this paper, according to the structural characteristics of bearings, the influence law of thermal-solid coupling effect on bearing structure is considered, and a novel transient temperature analysis model of damaged bearings is established. First, a quasi-static analysis of the bearing is performed to obtain the variation laws of the key parameters of the bearing under thermal expansion. Then, the load variation law of the bearing under the condition of damage is discussed, and the heat generation and heat transfer of the damaged bearing during operation are studied. Based on the thermal grid method, a transient temperature analysis model of the damaged bearing is developed. Finally, the model is tested experimentally and the influence of the rotate speed and load on the bearing temperature variation is analyzed. The results show that the established model can effectively predict the temperature variation and thermal equilibrium temperature of damaged bearings.

Femtomolar Biodetection by a Compact Core-Shell 3D Chiral Metamaterial.

Advanced sensing tools, detecting extremely low concentrations of circulating biomarkers, can open unexplored routes toward early diagnostics and diseases progression monitoring. Here, we demonstrate the sensing capabilities of a chip-based metamaterial, combining 3D chiral geometry with a functional core-shell nanoarchitecture. The chiral metamaterial provides a circular polarization-dependent optical response, allowing analysis in a complex environment without significant background interferences. The functional nanoarchitecture, based on the conformal coating with a polymer shell, modifies the chiral metamaterial near- and far-field optical response because of the energy transfer between dielectric shell polarization charges and plasmonic core free electrons, leading to efficient interaction with biomolecules. The system sensitivity slope is 27 nm/pM, in the detection of TAR DNA-binding protein 43, clinically relevant for neurodegenerative diseases. Measurements were performed in spiked solution and in human serum with concentrations from 1 pM down to 10 fM, which is a range not accessible with common immunological assays, opening new perspectives for next-generation biomedical systems.

Towards engineering ectomycorrhization into switchgrass bioenergy crops via a lectin receptor-like kinase.

Soil-borne microbes can establish compatible relationships with host plants, providing a large variety of nutritive and protective compounds in exchange for photosynthesized sugars. However, the molecular mechanisms mediating the establishment of these beneficial relationships remain unclear. Our previous genetic mapping and whole-genome resequencing studies identified a gene deletion event of a Populus trichocarpa lectin receptor-like kinase gene PtLecRLK1 in Populus deltoides that was associated with poor-root colonization by the ectomycorrhizal fungus Laccaria bicolor. By introducing PtLecRLK1 into a perennial grass known to be a non-host of L. bicolor, switchgrass (Panicum virgatum L.), we found that L. bicolor colonizes ZmUbipro-PtLecRLK1 transgenic switchgrass roots, which illustrates that the introduction of PtLecRLK1 has the potential to convert a non-host to a host of L. bicolor. Furthermore, transcriptomic and proteomic analyses on inoculated-transgenic switchgrass roots revealed genes/proteins overrepresented in the compatible interaction and underrepresented in the pathogenic defence pathway, consistent with the view that pathogenic defence response is down-regulated during compatible interaction. Metabolomic profiling revealed that root colonization in the transgenic switchgrass was associated with an increase in N-containing metabolites and a decrease in organic acids, sugars, and aromatic hydroxycinnamate conjugates, which are often seen in the early steps of establishing compatible interactions. These studies illustrate that PtLecRLK1 is able to render a plant susceptible to colonization by the ectomycorrhizal fungus L. bicolor and shed light on engineering mycorrhizal symbiosis into a non-host to enhance plant productivity and fitness on marginal lands.

Mapping and Validation of a Stable Quantitative Trait Locus Conferring Maize Resistance to Gibberella Ear Rot.

Gibberella ear rot (GER), a prevalent disease caused by Fusarium graminearum, can result in significant yield loss and carcinogenic mycotoxin contamination in maize worldwide. However, only a few quantitative trait loci (QTLs) for GER resistance have been reported. In this study, we evaluated a Chinese recombinant inbred line (RIL) population comprising 204 lines, developed from a cross between a resistant parent DH4866 and a susceptible line T877, in three field trials under artificial inoculation with F. graminearum. The RIL population and their parents were genotyped with an Affymetrix microarray CGMB56K SNP Array. Based on the genetic linkage map constructed using 1,868 bins as markers, 11 QTLs, including five stable QTLs, were identified by individual environment analysis. Joint multiple environments analysis and epistatic interaction analysis revealed six additive and six epistatic (additive × additive) QTLs, respectively. None of the QTLs could explain more than 10% of phenotypic variation, suggesting that multiple minor-effect QTLs contributed to the genetic component of resistance to GER, and both additive and epistatic effects contributed to the genetic architecture of resistance to GER. A novel QTL, qGER4.09, with the largest effect, identified and validated using 588 F2 individuals, was colocalized with genomic regions for Fusarium ear rot and Aspergillus ear rot, indicating that this genetic locus likely confers resistance to multiple pathogens and can potentially be utilized in breeding maize varieties aimed at improving the resistance not only to GER but also other ear rot diseases.

Genome-Wide Characterization of Jasmonates Signaling Components Reveals the Essential Role of ZmCOI1a-ZmJAZ15 Action Module in Regulating Maize Immunity to Gibberella Stalk Rot.

Gibberella stalk rot (GSR) by Fusarium graminearum causes significant losses of maize production worldwide. Jasmonates (JAs) have been broadly known in regulating defense against pathogens through the homeostasis of active JAs and COI-JAZ-MYC function module. However, the functions of different molecular species of JAs and COI-JAZ-MYC module in maize interactions with Fusarium graminearum and regulation of diverse metabolites remain unknown. In this study, we found that exogenous application of MeJA strongly enhanced resistance to GSR. RNA-seq analysis showed that MeJA activated multiple genes in JA pathways, which prompted us to perform a genome-wide screening of key JA signaling components in maize. Yeast Two-Hybrid, Split-Luciferase, and Pull-down assays revealed that the JA functional and structural mimic coronatine (COR) functions as an essential ligand to trigger the interaction between ZmCOIa and ZmJAZ15. By deploying CRISPR-cas9 knockout and Mutator insertional mutants, we demonstrated that coi1a mutant is more resistant, whereas jaz15 mutant is more susceptible to GSR. Moreover, JA-deficient opr7-5opr8-2 mutant displayed enhanced resistance to GSR compared to wild type. Together, these results provide strong evidence that ZmJAZ15 plays a pivotal role, whereas ZmCOIa and endogenous JA itself might function as susceptibility factors, in maize immunity to GSR.

Printed Nanochain-Based Colorimetric Assay for Quantitative Virus Detection.

Fast and ultrasensitive detection of pathogens is very important for efficient monitoring and prevention of viral infections. Here, we demonstrate a label-free optical detection approach that uses a printed nanochain assay for colorimetric quantitative testing of viruses. The antibody-modified nanochains have high activity and specificity which can rapidly identify target viruses directly from biofluids in 15 min, as well as differentiate their subtypes. Arising from the resonance induced near-field enhancement, the color of nanochains changes with the binding of viruses that are easily observed by a smartphone. We achieve the detection limit of 1 PFU µL-1 through optimizing the optical response of nanochains in visible region. Besides, it allows for real-time response to virus concentrations ranging from 0 to 1.0×105  PFU mL-1 . This low-cost and portable platform is also applicable to rapid detection of other biomarkers, making it attractive for many clinical applications.

Crystal structures of Babesia microti lactate dehydrogenase BmLDH reveal a critical role for Arg99 in catalysis.

The tick- and transfusion-transmitted human pathogen Babesia microti infects host erythrocytes to cause the pathologic symptoms associated with human babesiosis, an emerging disease with worldwide distribution and potentially fatal clinical outcome. Drugs currently recommended for the treatment of babesiosis are associated with a high failure rate and significant adverse events, highlighting the urgent need for more-effective and safer babesiosis therapies. Unlike other apicomplexan parasites, B. microti lacks a canonical lactate dehydrogenase (LDH) but instead expresses a unique enzyme, B. microti LDH (BmLDH), acquired through evolution by horizontal transfer from a mammalian host. Here, we report the crystal structures of BmLDH in apo state and ternary complex (enzyme-NADH-oxamate) solved at 2.79 and 1.89 Å. Analysis of these structures reveals that upon binding to the coenzyme and substrate, the active pocket of BmLDH undergoes a major conformational change from an opened and disordered to a closed and stabilized state. Biochemical assays using wild-type and mutant B. microti and human LDHs identified Arg99 as a critical residue for the catalytic activity of BmLDH but not its human counterpart. Interestingly, mutation of Arg99 to Ala had no impact on the overall structure and affinity of BmLDH to NADH but dramatically altered the closure of the enzyme's active pocket. Together, these structural and biochemical data highlight significant differences between B. microti and human LDH enzymes and suggest that BmLDH could be a suitable target for the development of selective antibabesial inhibitors.-Yu, L., Shen, Z., Liu, Q., Zhan, X., Luo, X., An, X., Sun, Y., Li, M., Wang, S., Nie, Z., Ao, Y., Zhao, Y., Peng, G., Ben Mamoun, C., He, L., Zhao, J. Crystal structures of Babesia microti lactate dehydrogenase BmLDH reveal a critical role for Arg99 in catalysis.

87 Sr/86 Sr isotope ratios in rocks determined using inductively coupled plasma tandem mass spectrometry in O2 mode without prior Sr purification.

RATIONALE: An inductively coupled plasma tandem mass spectrometry (ICP-MS/MS) instrument can be developed to determine 87 Sr/86 Sr ratios with an external precision better than 0.05% relative standard deviation (RSD) in "mass shift" mode without prior Sr purification. Previous studies suggested using CH3 F, N2 O, and SF6 as reaction gases for this method because a better reaction rate can be achieved with Sr+ than with O2 in the reaction cell. However, these gases are not commonly used in general chemistry laboratories, and processes using these gases are difficult to implement quickly due to regulations. We aim to develop a rapid method that can be applied to many samples for the accurate determination of 87 Sr/86 Sr isotope ratios with precision below 0.1% RSD (or approximately to the fourth decimal place). METHODS: We evaluated the accuracy and precision of 87 Sr/86 Sr ratios in certified reference materials and different rock types determined using ICP-MS/MS with O2 as the reaction gas in comparison with those determined using the multicollector inductively coupled plasma mass spectrometry (MC-ICP-MS) method. RESULTS: This study showed that by using the ICP-MS/MS method, the 87 Sr/86 Sr ratios of BCR-2 and BHVO-2 do not vary significantly with and without prior Sr purification; when the Sr concentration of the measured solution is within the range of 60-350 ng/mL, there is no significant effect on the measured 87 Sr/86 Sr ratios. The results also showed that the 87 Sr/86 Sr ratios of 23 different rock types measured by ICP-MS/MS and MC-ICP-MS methods agree very well. CONCLUSIONS: The precision of the 87 Sr/86 Sr ratio measured using ICP-MS/MS varies between 0.0001 and 0.0019 (2SD). This precision is less than that of the MC-ICP-MS method but is sufficient for certain applications, such as identifying 87 Sr/86 Sr ratios in different rock types. These results suggest that the developed ICP-MS/MS method has the potential for future studies involving the identification of Sr sources.

Characterization of the variable merozoite surface antigen (VMSA) gene family of Babesia orientalis.

Due to its wide presence in apicomplexan parasites as well as high polymorphism and antigenic diversity, the variable merozoite surface antigen (VMSA) family in Babesia sp. has attracted increasing attention of researchers. Here, all the reported VMSA genes of Babesia spp. were obtained from GenBank, and multiple alignments were performed by using conserved regions to blast the Babesia orientalis genome database (unpublished data). Five MSA genes (named MSA-2a1, MSA-2a2, MSA-2c1, MSA-1, and MSA-2c2, respectively) were identified, sequenced, and cloned from B. orientalis, which were shown to encode proteins with open reading frames ranging in size from 266 (MSA-2c1) to 317 (MSA-1) amino acids. All the five proteins contain an MSA-2c superfamily conserved domain, with an identical signal peptide and glycosyl phosphatidyl inositol (GPI)-anchor for each of them. The five proteins were also predicted to contain B cell epitopes, with only three for BoMSA-2c1, the smallest protein in the BoVMSA family, while at least six for each of the others. Notably, BoMSA-2a1 has 2 identical copies, a specific phenomenon only present in B. orientalis. This research has determined the MSA genes of B. orientalis and provides a genetic basis for further research of functional genes in B. orientalis.

Encapsulation of tartary buckwheat flavonoids and application to yoghurt.

Aims: The present work investigates the effect of tartary buckwheat flavonoid (TBF) capsules on the physical and chemical properties of yoghurt using polymeric whey protein (PWP) as a wall material.Methods: PWP was prepared by thermal polymerisation. TBF was encapsulated using PWP as the wall material via the pore-coagulation bath method. The physicochemical properties of the TBF capsules, such as the entrapment yield, moisture, average particle size, particle size distribution, surface morphology, molecular interactions, and thermal stability were investigated, in addition to the release of TBF in simulated gastric and intestinal juices. Yoghurt formulation was carried out using encapsulated TBF (3%, w/w), blank PWP beads (2.7%, w/w), and unencapsulated TBF (0.3%, w/w). A control yoghurt sample was prepared without these ingredients. The effects of encapsulated TBF on the chemical composition, acidity, texture, synaeresis, sensory properties, number of Streptococcus thermophilus and Lactobacillus, and other physical and chemical properties of the yoghurt were investigated.Results: TBF capsules were found to be sphere-shaped with porous surfaces, an average particle size of 1728.67 µm, an encapsulation yield of 92.85 ± 1.98% (w/w), and a glass transition temperature of 152.06 °C. When the TBF capsules were exposed to simulated gastric fluid for 4 h, the TBF release rate was 15.75% (w/w), while in simulated intestinal fluid, the TBF release rate reached 65.99% (w/w) after 1 h. After 5-6 h in simulated intestinal fluid, the TBF release rate reached 100% (w/w). The protein content of the yoghurt with encapsulated TBF was 3.57 ± 0.26% (w/w, p < 0.01), and the numbers of Lactobacillus and Streptococcus thermophilus were 2.45 ± 0.98 × 108 (p < 0.01) and 5.43 ± 2.24 × 107 CFU/mL (p < 0.05), respectively, with strong water retention being detected (p < 0.01). Samples containing the encapsulated TBF exhibited a significantly higher acceptability than the unencapsulated TBF (p < 0.01).Conclusions: Encapsulation using PWP effectively delivers TBF to the small intestine through the stomach. It also masks the bitter taste, enhances the colour of TBF-containing yoghurt, and improves the physical and chemical properties of the yoghurt.

Identification of Phytocyanin Gene Family in Legume Plants and their Involvement in Nodulation of Medicago truncatula.

The establishment of symbiosis between legume and rhizobium results in the formation of nodule. Phytocyanins (PCs) are a class of plant-specific blue copper proteins, playing critical roles in plant development including nodule formation. Although a few PC genes have been isolated from nodules, their functions are still unclear. Here, we performed a genome-wide identification of PC family in seven sequenced legume species (Medicago truncatula, Glycine max, Cicer arietinum, Cajanus cajan, Lotus japonicus, Vigna angularis and Phaseolus vulgaris) and found PCs experienced a remarkable expansion in M. truncatula and G. max. Further, we conducted an in-depth analysis of PC family in the model legume M. truncatula. Briefly, 82 MtPCs were divided into four subfamilies and clustered into seven clades, with a large proportion of tandem duplications and various cross-tissues expression patterns. Importantly, some PCs, such as MtPLC1, MtENODL27 and MtENODL28 were preferentially expressed in nodules. Further, RNA interference (RNAi) experiment revealed the knockdown of MtENDOL27 and MtENDOL28 impaired rhizobia infection, nodule numbers and nitrogenase activity. Moreover, in the MtENODL27-RNAi nodules, the infected cells were reduced and the symbiosomes did not reach the elongated stage, indicating MtENDOL27 is required for rhizobia infection and nodule development. In addition, co-expression analysis showed MtPLC1, MtENODL27 and MtENODL28 were grouped into two different functional modules and co-expressed with the known symbiotic nitrogen fixation-related genes, suggesting that they might participate in nodulation via different ways. In summary, this study provides a useful resource for future researches on the structure and function of PCs in nodulation.

N-TFA-Gly-Bt-Based Stereoselective Synthesis of Substituted 3-Amino Tetrahydro-2H-pyran-2-ones via an Organocatalyzed Cascade Process.

Chiral-substituted 3-amino tetrahydro-2H-pyran-2-ones were prepared in excellent enantioselectivities (up to 99% ee) via an organo-catalyzed cascade procedure with N-TFA-Gly-Bt and α,ß-unsaturated aldehydes as the substrates. The corresponding tetrahydro-2H-pyran-2-ones can be used for further synthetic transformations that furnish chiral-substituted 3-aminopiperidin-2-ones with high levels of stereoselectivity.

Cathepsin L-a novel cysteine protease from Haemaphysalis flava Neumann, 1897.

Ixodid ticks are ectoparasites responsible for the transmission of a large number of bacterial, viral, and protozoan pathogens to animals and humans. As long-term blood-pool feeders, the digestion of host blood is critical to their development as well as to the establishment of the sexual cycle of hemoparasites such as Babesia parasites, the agents of human and animal babesiosis. Previous studies have demonstrated that cysteine proteases are involved in blood digestion, embryogenesis, and pathogen transmission in other species of ticks, but their characteristics and functions are still unidentified in Haemaphysalis flava. Here, we describe the characterization of a cysteine protease HfCL from H. flava. We show that HfCL belongs to the L-like papain family of proteases, exhibits high expression in nymphs and adults, and localizes to both the midgut and salivary glands. Biochemical assays using purified recombinant enzyme reveal that rHfCL can hydrolyze the fluorogenic substrate Z-phe-Arg-MCA with optimal activity detected at pH 6. Furthermore, the short-term growth assay indicates that rHfCL can inhibit the intraerythrocytic development of Babesia microti and Babesia gibsoni in vitro.