Interregulation between fragile X mental retardation protein and methyl CpG binding protein 2 in the mouse posterior cerebral cortex.

Several X-linked neurodevelopmental disorders including Rett syndrome, induced by mutations in the MECP2 gene, and fragile X syndrome (FXS), caused by mutations in the FMR1 gene, share autism-related features. The mRNA coding for methyl CpG binding protein 2 (MeCP2) has previously been identified as a substrate for the mRNA-binding protein, fragile X mental retardation protein (FMRP), which is silenced in FXS. Here, we report a homeostatic relationship between these two key regulators of gene expression in mouse models of FXS (Fmr1 Knockout (KO)) and Rett syndrome (MeCP2 KO). We found that the level of MeCP2 protein in the cerebral cortex was elevated in Fmr1 KO mice, whereas MeCP2 KO mice displayed reduced levels of FMRP, implicating interplay between the activities of MeCP2 and FMRP. Indeed, knockdown of MeCP2 with short hairpin RNAs led to a reduction of FMRP in mouse Neuro2A and in human HEK-293 cells, suggesting a reciprocal coupling in the expression level of these two regulatory proteins. Intra-cerebroventricular injection of an adeno-associated viral vector coding for FMRP led to a concomitant reduction in MeCP2 expression in vivo and partially corrected locomotor hyperactivity. Additionally, the level of MeCP2 in the posterior cortex correlated with the severity of the hyperactive phenotype in Fmr1 KO mice. These results demonstrate that MeCP2 and FMRP operate within a previously undefined homeostatic relationship. Our findings also suggest that MeCP2 overexpression in Fmr1 KO mouse posterior cerebral cortex may contribute to the fragile X locomotor hyperactivity phenotype.

Relationship between increased systemic immune-inflammation index and coronary slow flow phenomenon.

BACKGROUND: Systemic immune-inflammation index (SII, platelet × neutrophil/lymphocyte ratio), a new marker of inflammation, is associated with adverse cardiovascular events, but its relationship with coronary slow flow phenomenon (CSFP) is unclear. Therefore, we aimed to investigate the relationship between SII and CSFP. METHODS: We enrolled consecutive patients who presented with chest pain, with normal/near-normal coronary angiography findings (n = 89 as CSFP group; n = 167 as control group). The baseline characteristics, laboratory parameters and angiographic characteristics of the two groups were compared. RESULTS: SII levels were significantly higher in the CSFP group than in the control group (409.7 ± 17.7 vs. 396.7 ± 12.7, p < 0.001). A significant positive correlation between SII and the mean thrombolysis in myocardial infarction frame count (mTFC) was found (r = 0.624, p < 0.001). SII increased with the number of coronary arteries involved in CSFP. In multivariate logistic regression analysis, SII/10 was an independent predictor of CSFP (odds ratio: 1.739, p < 0.001). In addition, the SII level > 404.29 was a predictor of CSFP with 67.4% sensitivity and 71.9% specificity. CONCLUSIONS: SII can predict the occurrence of CSFP.

Bacteria and cancer cell pearl chain under dielectrophoresis.

In this work, we aim to observe and study the physics of bacteria and cancer cells pearl chain formation under dielectrophoresis (DEP). Experimentally, we visualized the formation of Bacillus subtilis bacterial pearl chain and human breast cancer cell (MCF-7) chain under positive and negative dielectrophoretic force, respectively. Through a simple simulation with creeping flow, AC/DC electric fields, and particle tracing modules in COMSOL, we examined the mechanism by which bacteria self-organize into a pearl chain across the gap between two electrodes via DEP. Our simulation results reveal that the region of greatest positive DEP force shifts from the electrode edge to the leading edge of the pearl chain, thus guiding the trajectories of free-flowing particles toward the leading edge via positive DEP. Our findings additionally highlight the mechanism why the free-flowing particles are more likely to join the existing pearl chain rather than starting a new pearl chain. This phenomenon is primarily due to the increase in magnitude of electric field gradient, and hence DEP force exerted, with the shortening gap between the pearl chain leading edge and the adjacent electrode. The findings shed light on the observed behavior of preferential pearl chain formation across electrode gaps.

Characterization of Shrink Film Properties for Rapid Microfluidics Lab-on-Chip Fabrication.

Shrink film is a thin sheet of polystyrene plastic that shrinks to 25-40% of its original size when heated. This study investigated the shrinkage factor of the film at different temperatures and baking times to determine the optimal fabrication recipe for shrink film microfluidic device production. Additionally, this study characterized the properties of shrink film, including minimum possible feature size and cross-section geometries, using manual engraving and the CAMEO 4 automated cutting machine. The optimal shrinkage factor ranged from 1.7 to 2.9 at 150 °C and a baking time of 4 min, producing the ideal size for microfluidic device fabrication. The X- and Y-axes shrank ~2.5 times, while Z-axis thickened by a factor of ~5.8 times. This study achieved a minimum feature size of 200 microns, limited by the collapsing of channel sidewalls when shrunk, leading to blockages in the microchannel. These findings demonstrate the feasibility and versatility of using shrink film as a cost-effective and efficient material for the rapid fabrication of microfluidic devices. The potential applications of this material in various fields such as the medical and biomedical industries, bacteria and algae culture and enumeration are noteworthy.

Urine osmolality assessment through the integration of urea hydrolysis and impedance measurement.

We present the development and validation of an impedance-based urine osmometer for accurate and portable measurement of urine osmolality. The urine osmolality of a urine sample can be estimated by determining the concentrations of the conductive solutes and urea, which make up approximately 94% of the urine composition. Our method utilizes impedance measurements to determine the conductive solutes and urea after hydrolysis with urease enzyme. We built an impedance model using sodium chloride (NaCl) and urea at various known concentrations. In this work, we validated the accuracy of the impedance-based urine osmometer by developing a proof-of-concept first prototype and an integrated urine dipstick second prototype, where both prototypes exhibit an average accuracy of 95.5 ± 2.4% and 89.9 ± 9.1%, respectively in comparison to a clinical freezing point osmometer in the hospital laboratory. While the integrated dipstick design exhibited a slightly lower accuracy than the first prototype, it eliminated the need for pre-mixing or manual pipetting. Impedance calibration curves for conductive and non-conductive solutes consistently yielded results for NaCl but underscored challenges in achieving uniform urease enzyme coating on the dipstick. We also investigated the impact of storing urine at room temperature for 24 hours, demonstrating negligible differences in osmolality values. Overall, our impedance-based urine osmometer presents a promising tool for point-of-care urine osmolality measurements, addressing the demand for a portable, accurate, and user-friendly device with potential applications in clinical and home settings.

ExtraCorporeal Membrane Oxygenation in the therapy for REfractory Septic shock with Cardiac function Under Estimated (ECMO-RESCUE): study protocol for a prospective, multicentre, non-randomised cohort study.

INTRODUCTION: Severe septic cardiomyopathy (SCM) is one of the main causes of refractory septic shock (RSS), with a high mortality. The application of venoarterial extracorporeal membrane oxygenation (ECMO) to support the impaired cardiac function in patients with septic shock remains controversial. Moreover, no prospective studies have been taken to address whether venoarterial ECMO treatment could improve the outcome of patients with sepsis-induced cardiogenic shock. The objective of this study is to assess whether venoarterial ECMO treatment can improve the 30-day survival rate of patients with sepsis-induced refractory cardiogenic shock. METHODS AND ANALYSIS: ExtraCorporeal Membrane Oxygenation in the therapy for REfractory Septic shock with Cardiac function Under Estimated is a prospective, multicentre, non-randomised, cohort study on the application of ECMO in SCM. At least 64 patients with SCM and RSS will be enrolled in an estimated ratio of 1:1.5. Participants taking venoarterial ECMO during the period of study are referred to as cohort 1, and patients receiving only conventional therapy without ECMO belong to cohort 2. The primary outcome is survival in a 30-day follow-up period. Other end points include survival to intensive care unit (ICU) discharge, hospital survival, 6-month survival, quality of life for long-term survival (EQ-5D score), successful rate of ECMO weaning, long-term survivors' cardiac function, the number of days alive without continuous renal replacement therapy, mechanical ventilation and vasopressor, ICU and hospital length of stay, the rate of complications potentially related to ECMO treatment. ETHICS AND DISSEMINATION: The trial has been approved by the Clinical Research and Application Institutional Review Board of the Second Affiliated Hospital of Guangzhou Medical University (2020-hs-51). Participants will be screened and enrolled from ICU patients with septic shock by clinicians, with no public advertisement for recruitment. Results will be disseminated in research journals and through conference presentations. TRIAL REGISTRATION NUMBER: NCT05184296.

Gefitinib (an EGFR tyrosine kinase inhibitor) plus anlotinib (an multikinase inhibitor) for untreated, EGFR-mutated, advanced non-small cell lung cancer (FL-ALTER): a multicenter phase III trial.

Dual inhibition of vascular endothelial growth factor and epidermal growth factor receptor (EGFR) signaling pathways offers the prospect of improving the effectiveness of EFGR-targeted therapy. In this phase 3 study (ClinicalTrial.gov: NCT04028778), 315 patients with treatment-naïve, EGFR-mutated, advanced non-small cell lung cancer (NSCLC) were randomized (1:1) to receive anlotinib or placebo plus gefitinib once daily on days 1-14 per a 3-week cycle. At the prespecified final analysis of progression-free survival (PFS), a significant improvement in PFS was observed for the anlotinib arm over the placebo arm (hazards ratio [HR] = 0.64, 95% CI, 0.48-0.80, P = 0.003). Particularly, patients with brain metastasis and those harboring EGFR amplification or high tumor mutation load gained significant more benefits in PFS from gefitinib plus anlotinib. The incidence of grade 3 or higher treatment-emergent adverse events was 49.7% of the patients receiving gefitinib plus anlotinib versus 31.0% of the patients receiving gefitinib plus placebo. Anlotinib plus gefitinib significantly improves PFS in patients with treatment-naïve, EGFR-mutated, advanced NSCLC, with a manageable safety profile.

[Effects of curcumin intake on kidney and liver pathological changes in T2DM rats].

OBJECTIVE: To investigate whether curcumin intake could improve kidney, liver pathological changes in type 2 diabetes (T2DM) rats. METHODS: 100 male Wistar rats were randomly divided into two groups: 10 rats in the control group; 90 in the T2DM model rats, the using low-dose treptozotocin (30 mg/kg BW) combined high sugar and high fat diet to induce T2DM model. After the success of the model induction, 39 T2DM rats met the selection criteria, which were randomly divided into 4 groups: T2DM model control group, low-dose curcumin group (50 mg/kg BW), curcumin dose group (150 mg/kg BW) and curcumin high-dose group (250 mg/kg BW), given intervention. After 45 days treatment, rats from each group were randomly selected four for pathological testing and observation of kidney and liver changes. RESULTS: Compared with the control group, the results showed that blood glucose and lipids in T2DM model group were significantly increased (P < 0.05). Compared to the T2DM control group, curcumin treatment significant improved kidney and liver pathological changes. CONCLUSION: Curcumin can improve liver and kidney pathological changes in T2DM rats.

A designer peptide against the EAG2-Kvß2 potassium channel targets the interaction of cancer cells and neurons to treat glioblastoma.

Glioblastoma (GBM) is an incurable brain cancer that lacks effective therapies. Here we show that EAG2 and Kvß2, which are predominantly expressed by GBM cells at the tumor-brain interface, physically interact to form a potassium channel complex due to a GBM-enriched Kvß2 isoform. In GBM cells, EAG2 localizes at neuron-contacting regions in a Kvß2-dependent manner. Genetic knockdown of the EAG2-Kvß2 complex decreases calcium transients of GBM cells, suppresses tumor growth and invasion and extends the survival of tumor-bearing mice. We engineered a designer peptide to disrupt EAG2-Kvß2 interaction, thereby mitigating tumor growth in patient-derived xenograft and syngeneic mouse models across GBM subtypes without overt toxicity. Neurons upregulate chemoresistant genes in GBM cells in an EAG2-Kvß2-dependent manner. The designer peptide targets neuron-associated GBM cells and possesses robust efficacy in treating temozolomide-resistant GBM. Our findings may lead to the next-generation therapeutic agent to benefit patients with GBM.

A Technique for Rapid Bacterial-Density Enumeration through Membrane Filtration and Differential Pressure Measurements.

In this article, we present a microfluidic technique for the rapid enumeration of bacterial density with a syringe filter to trap bacteria and the quantification of the bacterial density through pressure difference measurement across the membrane. First, we established the baseline differential pressure and hydraulic resistance for a filtration membrane by fully wetting the filter with DI water. Subsequently, when bacteria were infused and trapped at the pores of the membrane, the differential pressure and hydraulic resistance also increased. We characterized the infusion time required for the bacterial sample to achieve a normalized hydraulic resistance of 1.5. An equivalent electric-circuit model and calibration data sets from parametric studies were used to determine the general form of a calibration curve for the prediction of the bacterial density of a bacterial sample. As a proof of concept, we demonstrated through blind tests with Escherichia coli that the device is capable of determining the bacterial density of a sample ranging from 7.3 × 106 to 2.2 × 108 CFU/mL with mean and median accuracies of 87.21% and 91.33%, respectively. The sample-to-result time is 19 min for a sample with lower detection threshold, while for higher-bacterial-density samples the measurement time is further shortened to merely 8 min.

Disposable flow cytometer with high efficiency in particle counting and sizing using an optofluidic lens.

Flow cytometers are widely applied to environmental monitoring, industrial testing, and biochemical studies. Integrating a flow cytometer into microfluidic networks helps to miniaturize the system and make it portable for field use. The integration of optical components, such as lenses, further improves the compactness and thus has been intensively studied recently. However, the current designs suffer from severe light scattering due to the roughness of the solid-based lens interface. In this Letter, we propose a flow cytometer using an optofluidic lens to focus the light beam. Benefiting from the smooth liquid-liquid lens interface and the refractive-index matching liquid as cladding streams, a light beam can be well focused without scattering. The variations of the signal peak values are reduced, owing to the small beam width at the beam waist. The device presents an efficient and accurate performance on both the counting and sizing of particles.

[Oleanolic acid-stimulated lipolysis in primary adipocytes and its mechanisms].

OBJECTIVE: To investigate the effect and mechanism of oleanolic acid (OA)-stimulated lipolysis in primary adipocytes. METHODS: Adipocytes were isolated from the epididymal fat pads of SD rats. The non-cytotoxic dosage of OA for primary adipocytes was established by MTT test. Adipocytes were divided into four groups: a control group and 3 OA-treated groups (25, 50 and 100 micromol/L). Glycerol released in the culture medium of the control and three OA-treated adipocyte groups was determined after incubation with OA for 4 and 24 hours. The glycerol released in the culture medium of three OA-treated adipocyte groups with protein kinase A inhibitor H-89 (10 micromol/L) was also determined. The expression of native perilipin and hormone-sensitive lipase (HSL) translocation were examined by immunoblotting method. RESULTS: Cytotoxicity was observed when the concentration of OA was 400 micromol/L. After incubation for 4 hours, the release of glycerol in the 50 micromol/L and 100 micromol/L OA groups was increased 12% and 20% (P < 0.05) respectively. After incubation for 24 hours, the release of glycerol was increased 14% (P > 0.05) and 23% (P < 0.05) respectively. There is no change of glycerol release in the three OA- and H-89-treated adipocyte groups. OA may down-regulate the expression of perilipin and promote HSL translocation. CONCLUSION: OA may stimulate the release of glycerol from primary adipocytes, and the cellular mechanisms of lipolytic action may involve in augmenting perilipin expression and promoting HSL translocation.

[Induction of injury to endothelium of pulmonary artery due to entero superantigen by up regulation of lymphocyte chemokine receptor 5].

OBJECTIVE: To observe the injurious effect of T cell activated by Staphylococcus enterotoxin B (SEB) on human pulmonary artery endothelial cell (HPAEC) and explore its possible mechanism. METHODS: HPAEC was cocultured with SEB-activated T cells supernatant, and the secretion of chemotactic factors from HPAEC was examined. The Transwell inserts was used in chemoattraction assays. After HPAECs were cocultured with T cells and 10 ng/ml SEB for 3 days, HPAEC damage was monitored by microscopy and the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay. RESULTS: Three kinds of tested chemokines showed a time dependent increase in all supernatant of HPAEC incubated with different concentrations of T cells. After 72 hours, the monocyte chemoattractant protein 1 (MCP 1, ng/ml) in 1×10(-2) , 1×10(-1), 1×10(0) T cell supernatant groups was 1.240±0.103, 4.200±0.305, 6.500±0.500, respectively, macrophage inflammatory protein 1α (MIP 1α, ng/ml) was 0.210±0.015, 0.287±0.012, 0.531±0.037, respectively , and Rantes (ng/ml) was 1.420±0.074, 7.634±0.630, 15.700±1.300, respectively. Rantes presented a two phase secretion mode: in early 6 hours it increased swiftly, but relatively slow at 12, 24, 48, 72 hours. T cell adherent to polycarbonate membrane increased after SEB stimulation in superantigen group compared with control group without SEB stimulation (86.38±14.50 vs. 16.50±2.50, P<0.01). When 10 ng/ml SEB-activated T cell was cocultured with HPAEC, more of originally suspended cultured T cells adhered to HPAEC monolayer [(15.50±1.08)% vs. (1.60±0.22)%, PP<0.01], whereas the cell adhesion ratio decreased markedly in 1 µg/ml Met Rantes group [(4.39±0.66)%, PP<0.01). FACs test of HPAEC adherent T cell showed lymphocyte chemokine receptor 5 (CCR5)/CD4 and CCR5/CD8 increased over 2.5 folds and 2.8 folds compared with 100 ng/ml SEB-activated T cell. Cell death rate of HPAEC was increased when cocultured with SEB-activated T cell in superantigen group compared with HPAEC normal incubation group [(32.50±4.50)% vs. (3.50±0.50)%, P<0.01]. CONCLUSION: Increased chemoattraction and adherence of SEB-activated T cells to HPAEC could damage HPAEC; this effect was possibly due to up regulation of CCR5 on T cell.

Dielectrophoretic trapping and impedance detection of Escherichia coli, Vibrio cholera, and Enterococci bacteria.

In this work, a dielectrophoretic impedance measurement (DEPIM) lab-on-chip device for bacteria trapping and detection of Escherichia coli, Vibrio cholerae, and Enterococcus is presented. Through the integration of SU-8 negative photoresist as a microchannel and the precise alignment of the SU-8 microchannel with the on-chip gold interdigitated microelectrodes, bacteria trapping efficiencies of up to 97.4%, 97.7%, and 37.7% were achieved for E. coli, V. cholerae, and Enterococcus, respectively. The DEPIM device enables a high detection sensitivity, which requires only a total number of 69 ± 33 E. coli cells, 9 ± 2 Vibrio cholera cells, and 36 ± 13 Enterococcus cells to observe a discernible change in system impedance for detection. Nonetheless, the corrected limit of detection for Enterococcus is 95 ± 34 after taking into consideration the lower trapping efficiency. In addition, a theoretical model is developed to allow for the direct estimation of the number of bacteria through a linear relationship with the change in the reciprocal of the overall system absolute impedance.

Accessory-free quantitative smartphone imaging of colorimetric paper-based assays.

The combination of smartphone technology and colorimetric paper-based microfluidics can enable simple, inexpensive diagnostics. However, imaging colorimetric diagnostic results via smartphones currently requires accessories to mitigate the influence of variability in surrounding lighting conditions. Here, we present an accessory-free smartphone-based colorimetric imaging method that enlists the built-in LED light source to dominate ambient lighting in combination with background and colour rescaling. This simple approach enables quantitative measurements from paper-based tests by compensating for different environmental lighting conditions and is universally applicable with respect to phone models and manufacturers. We demonstrate the method with three dominant phone makes and models in a cell counting application with a paper-based yeast detection device. The detection results are in good agreement with cell counting using automated cell counters. Eliminating the need for make/model specific accessories, this approach helps realize the potential for low-cost, broadly applicable paper-based diagnostics.

Two-dimensional planar swimming selects for high DNA integrity sperm.

Selection of high-quality sperm is critical to the success of assisted reproductive technologies. Clinical screening for top sperm has long focused on sperm swimming ability when following boundaries or when fully free of constraints. In this work, we demonstrate a sperm selection approach with parallel 2 µm tall confined selection channels that prohibit rotation of the sperm head and require planar swimming. We demonstrate that a planar swimming subpopulation of sperm capable of entering and navigating these channels has DNA integrity superior to the freely-swimming motile or raw sperm populations over a wide range of semen sample qualities. The DNA integrity of the selected sperm was significantly higher than that of the corresponding raw samples for donor samples and clinical patient samples, respectively. In side-by-side testing, this method outperforms current clinical selection methods, density gradient centrifugation and swim-up, as well as sperm selected via general motility. Planar swimming represents a viable sperm selection mechanism with the potential to improve outcomes for couples and offspring.

Prediction of DNA Integrity from Morphological Parameters Using a Single-Sperm DNA Fragmentation Index Assay.

Intracytoplasmic sperm injection is a popular form of in vitro fertilization, where single sperm are selected by a clinician and injected into an egg. Whereas clinicians employ general morphology-based guidelines to select the healthiest-looking sperm, it remains unclear to what extent an individual sperm's physical parameters correlate with the quality of internal DNA cargo-a measurement that cannot be obtained without first damaging the sperm. Herein, a single-cell DNA fragmentation index (DFI) assay is demonstrated, which combines the single-cell nature of the acridine orange test with the quantitative aspect of the sperm chromatin structure assay, to create a database of DFI-scored brightfield images. Two regression predictive models, linear and nonlinear regression, are used to quantify the correlations between individual sperm morphological parameters and DFI score (with model test r at 0.558 and 0.620 for linear and nonlinear regression models, respectively). The sample is also split into two categories of either relatively good or bad DFIs and a classification predictive model based on logistic regression is used to categorize sperm, resulting in a test accuracy of 0.827. Here, the first systematic study is presented on the correlation and prediction of sperm DNA integrity from morphological parameters at the single-cell level.

Deep learning-based selection of human sperm with high DNA integrity.

Despite the importance of sperm DNA to human reproduction, currently no method exists to assess individual sperm DNA quality prior to clinical selection. Traditionally, skilled clinicians select sperm based on a variety of morphological and motility criteria, but without direct knowledge of their DNA cargo. Here, we show how a deep convolutional neural network can be trained on a collection of ~1000 sperm cells of known DNA quality, to predict DNA quality from brightfield images alone. Our results demonstrate moderate correlation (bivariate correlation ~0.43) between a sperm cell image and DNA quality and the ability to identify higher DNA integrity cells relative to the median. This deep learning selection process is directly compatible with current, manual microscopy-based sperm selection and could assist clinicians, by providing rapid DNA quality predictions (under 10 ms per cell) and sperm selection within the 86th percentile from a given sample.

Low pressure supercritical CO2 extraction of astaxanthin from Haematococcus pluvialis demonstrated on a microfluidic chip.

This study demonstrates the efficacy of low pressure supercritical CO2 extraction of astaxanthin from disrupted Haematococcus pluvialis. A microfluidic reactor was employed that enabled excellent control and allowed direct monitoring of the whole process at the single cell level, in real time. Astaxanthin extraction using ScCO2 achieved 92% recovery at 55 °C and 8 MPa applied over 15 h. With the addition of co-solvents, ethanol and olive oil, the extraction rates in both experiments were significantly improved reaching full recovery within a few minutes. Notably, for the ethanol case, the timescales of extraction process are reduced 1800-fold from 15 h to 30 s at 55 °C and 8 MPa, representing the fastest complete astaxanthin extraction at such low pressures.

Biophysical and structural characterization of the thermostable WD40 domain of a prokaryotic protein, Thermomonospora curvata PkwA.

WD40 proteins belong to a big protein family with members identified in every eukaryotic proteome. However, WD40 proteins were only reported in a few prokaryotic proteomes. Using WDSP ( http://wu.scbb.pkusz.edu.cn/wdsp/ ), a prediction tool, we identified thousands of prokaryotic WD40 proteins, among which few proteins have been biochemically characterized. As shown in our previous bioinformatics study, a large proportion of prokaryotic WD40 proteins have higher intramolecular sequence identity among repeats and more hydrogen networks, which may indicate better stability than eukaryotic WD40s. Here we report our biophysical and structural study on the WD40 domain of PkwA from Thermomonospora curvata (referred as tPkwA-C). We demonstrated that the stability of thermophilic tPkwA-C correlated to ionic strength and tPkwA-C exhibited fully reversible unfolding under different denaturing conditions. Therefore, the folding kinetics was also studied through stopped-flow circular dichroism spectra. The crystal structure of tPkwA-C was further resolved and shed light on the key factors that stabilize its beta-propeller structure. Like other WD40 proteins, DHSW tetrad has a significant impact on the stability of tPkwA-C. Considering its unique features, we proposed that tPkwA-C should be a great structural template for protein engineering to study key residues involved in protein-protein interaction of a WD40 protein.