Remodelling of the glycome of B-cell precursor acute lymphoblastic leukemia cells developing drug-tolerance.

Reduced responsiveness of precursor B-acute lymphoblastic leukemia (BCP-ALL) to chemotherapy can be first detected in the form of minimal residual disease leukemia cells that persist after 28 days of initial treatment. The ability of these cells to resist chemotherapy is partly due to the microenvironment of the bone marrow, which promotes leukemia cell growth and provides protection, particularly under these conditions of stress. It is unknown if and how the glycocalyx of such cells is remodelled during the development of tolerance to drug treatment, even though glycosylation is the most abundant cell surface post-translational modification present on the plasma membrane. To investigate this, we performed omics analysis of BCP-ALL cells that survived a 30-day vincristine chemotherapy treatment while in co-culture with bone marrow stromal cells. Proteomics showed decreased levels of some metabolic enzymes. Overall glycocalyx changes included a shift from Core-2 to less complex Core-1 O-glycans, and reduced overall sialylation, with a shift from α2-6 to α2-3 linked Neu5Ac. Interestingly, there was a clear increase in bisecting complex N-glycans with a concomitant increased mRNA expression of MGAT3 , the only enzyme known to form bisecting N-glycans. These small but reproducible quantitative differences suggest that individual glycoproteins become differentially glycosylated. Glycoproteomics confirmed glycosite-specific modulation of cell surface and lysosomal proteins in drug-tolerant BCP-ALL cells, including HLA-DRA, CD38, LAMP1 and PPT1. We conclude that drug-tolerant persister leukemia cells that grow under continuous chemotherapy stress have characteristic glycotraits that correlate with and perhaps contribute to their ability to survive and could be tested as neoantigens in drug-resistant leukemia.

Activation mechanisms of dimeric mechanosensitive OSCA/TMEM63 channels.

OSCA/TMEM63 channels, which have transporter-like architectures, are bona fide mechanosensitive (MS) ion channels that sense high-threshold mechanical forces in eukaryotic cells. The activation mechanism of these transporter-like channels is not fully understood. Here we report cryo-EM structures of a dimeric OSCA/TMEM63 pore mutant OSCA1.1-F516A with a sequentially extracellular dilated pore in a detergent environment. These structures suggest that the extracellular pore sequential dilation resembles a flower blooming and couples to a sequential contraction of each monomer subunit towards the dimer interface and subsequent extrusion of the dimer interface lipids. Interestingly, while OSCA1.1-F516A remains non-conducting in the native lipid environment, it can be directly activated by lyso-phosphatidylcholine (Lyso-PC) with reduced single-channel conductance. Structural analysis of OSCA1.1-F516A in lyso-PC-free and lyso-PC-containing lipid nanodiscs indicates that lyso-PC induces intracellular pore dilation by attracting the M6b to upward movement away from the intracellular side thus extending the intracellular pore. Further functional studies indicate that full activation of MS OSCA/TMEM63 dimeric channels by high-threshold mechanical force also involves the opening of both intercellular and extracellular pores. Our results provide the fundamental activation paradigm of the unique transporter-like MS OSCA/TMEM63 channels, which is likely applicable to functional branches of the TMEM63/TMEM16/TMC superfamilies.

Channelrhodopsins with distinct chromophores and binding patterns.

Channelrhodopsins are popular optogenetic tools in neuroscience, but remain poorly understood mechanistically. Here we report the cryo-EM structures of channelrhodopsin-2 (ChR2) from Chlamydomonas reinhardtii and H. catenoides kalium channelrhodopsin (KCR1). We show that ChR2 recruits an endogenous N-retinylidene-PE-like molecule to a previously unidentified lateral retinal binding pocket, exhibiting a reduced light response in HEK293 cells. In contrast, H. catenoides kalium channelrhodopsin (KCR1) binds an endogenous retinal in its canonical retinal binding pocket under identical condition. However, exogenous ATR reduces the photocurrent magnitude of wild type KCR1 and also inhibits its leaky mutant C110T. Our results uncover diverse retinal chromophores with distinct binding patterns for channelrhodopsins in mammalian cells, which may further inspire next generation optogenetics for complex tasks such as cell fate control.

Quantitative bias analysis methods for summary level epidemiologic data in the peer-reviewed literature: a systematic review.

OBJECTIVE: Quantitative bias analysis (QBA) methods evaluate the impact of biases arising from systematic errors on observational study results. This systematic review aimed to summarize the range and characteristics of quantitative bias analysis (QBA) methods for summary level data published in the peer-reviewed literature. STUDY DESIGN AND SETTING: We searched MEDLINE, Embase, Scopus, and Web of Science for English-language articles describing QBA methods. For each QBA method, we recorded key characteristics, including applicable study designs, bias(es) addressed; bias parameters, and publicly available software. The study protocol was pre-registered on the Open Science Framework (https://osf.io/ue6vm/). RESULTS: Our search identified 10,249 records, of which 53 were articles describing 57 QBA methods for summary level data. Of the 57 QBA methods, 53 (93%) were explicitly designed for observational studies, and 4 (7%) for meta-analyses. There were 29 (51%) QBA methods that addressed unmeasured confounding, 19 (33%) misclassification bias, 6 (11%) selection bias, and 3 (5%) multiple biases. 38 (67%) QBA methods were designed to generate bias-adjusted effect estimates and 18 (32%) were designed to describe how bias could explain away observed findings. 22 (39%) articles provided code or online tools to implement the QBA methods. CONCLUSION: In this systematic review, we identified a total of 57 QBA methods for summary level epidemiologic data published in the peer-reviewed literature. Future investigators can use this systematic review to identify different QBA methods for summary level epidemiologic data.

Assessment of central nervous system vasculitis in children based on high-resolution vascular wall imaging.

Objectives: Central nervous system vasculitis (CNSV) is a rare disease. High-resolution vessel wall imaging (HR-VWI) enables the identification of inflammatory changes within the vessel wall. Few studies have applied HR-VWI to assess CNSV in children. This study delves into the utility of HR-VWI for diagnosing and treating CNSV in children, with the aim of enhancing clinical diagnosis and efficacy evaluation. Methods: Imaging data were acquired from children who underwent HR-VWI examinations. The study meticulously analysed clinical data and laboratory tests to discern the characteristics and distribution patterns of diverse vasculitis forms. Results: In children, CNSV mainly involves medium vessels with grade 1 and 2 stenosis (grade 4 stenosis is rare), and the imaging features generally show centripetal and moderate enhancement, suggesting that this feature is specific for the diagnosis of CNSV. High-grade stenosis, concentric enhancement and strong enhancement of the vasculature indicate more severe disease activity. Remarkably, HR-VWI proved to be significantly more sensitive than magnetic resonance angiography in detecting CNSV. Among the 13 cases subjected to imaging review, 8 demonstrated a reduction or resolution of vessel wall inflammation. In contrast, five patients exhibited worsening inflammation in the vessel wall. HR-VWI demonstrated that changes in vessel wall inflammation were closely correlated with changes in brain parenchymal lesions and symptoms. Conclusion: This study underscores the diagnostic value of HR-VWI in CNSV assessment and treatment monitoring, offering a quantitative evaluation of CNSV in children.

Mechanosensitive channel MscL gating transitions coupling with constriction point shift.

The mechanosensitive channel of large conductance (MscL) acts as an "emergency release valve" that protects bacterial cells from acute hypoosmotic stress, and it serves as a paradigm for studying the mechanism underlying the transduction of mechanical forces. MscL gating is proposed to initiate with an expansion without opening, followed by subsequent pore opening via a number of intermediate substates, and ends in a full opening. However, the details of gating process are still largely unknown. Using in vivo viability assay, single channel patch clamp recording, cysteine cross-linking, and tryptophan fluorescence quenching approach, we identified and characterized MscL mutants with different occupancies of constriction region in the pore domain. The results demonstrated the shifts of constriction point along the gating pathway towards cytoplasic side from residue G26, though G22, to L19 upon gating, indicating the closed-expanded transitions coupling of the expansion of tightly packed hydrophobic constriction region to conduct the initial ion permeation in response to the membrane tension. Furthermore, these transitions were regulated by the hydrophobic and lipidic interaction with the constricting "hot spots". Our data reveal a new resolution of the transitions from the closed to the opening substate of MscL, providing insights into the gating mechanisms of MscL.

Excavation method optimization and mechanical responses investigating of a shallow buried super large section tunnels: a case study in Zhejiang.

The construction of super large section (SLS) shallow buried tunnels involves challenges related to their large span, high flat rate, and complex construction process. Selecting an appropriate excavation method is crucial for ensuring stability, controlling costs, and managing the construction timeline. This study focuses on the selection of excavation methods and the mechanical responses of SLS tunnels in different types of surrounding rock. The research is based on the Yangjiashan tunnel project in Zhejiang Province, China, which is a four-line highway tunnel with a span of 21.3 m. Three sequential excavation methods were proposed and simulated using the three-dimensional finite difference method: the "upper first and lower later" side drift (SD) method, the central diaphragm method, and the top heading and bench (HB) method. The mechanical response characteristics of tunnel construction under these methods were investigated, including rock deformation, rock pressure, and the internal forces acting on the primary support. By comparing the performance of the three construction methods in rock masses of Grades III to V, the study aimed to determine the optimal construction method for SLS tunnels considering factors such as safety, cost, and schedule. Field tests were conducted to evaluate the effectiveness of the optimized construction scheme. The results of the field monitoring indicated that the "upper first and lower later" SD method in Grade V rock mass and the HB method in Grade III to IV rock mass are feasible and cost-effective under certain conditions. The research findings provide valuable insights for the design and construction of SLS tunnels in complex conditions, serving as a reference for engineers and project managers.

Development and validation of a major adverse limb events prediction model for peripheral arterial disease with frailty.

OBJECTIVE: To investigate the risk factors for major limb adverse events (MALE) in peripheral arterial disease (PAD) combined with frailty and to develop and validate a risk prediction model of MALE. METHODS: This prospective study was performed in the vascular surgery department of patients in six hospitals in southwest China. Prospective collection of patients with PAD combined with frailty from February 1 to December 20, 2021, with MALE as the primary outcome, and followed for 1 year. The cohort was divided into a development cohort and a validation cohort. In the development cohort, a multivariate risk prediction model was developed to predict MALE using random forests for variable selection and multivariable Cox regression analysis. The model is represented by a visualized nomogram and a web-based calculator. The model performance was tested with the validation cohort and assessed using the C-statistic and calibration plots. RESULTS: A total of 1179 patients were prospectively enrolled from February 1 to December 20, 2021. Among 816 patients with PAD who were included in the analysis, the median follow-up period for this study was 9 ± 4.07 months, the mean age was 74.64 ± 9.43 years, and 249 (30.5%) were women. Within 1 year, 222 patients (27.2%) developed MALE. Target lesion revascularizations were performed in 99 patients (12.1%), and amputations were performed in 131 patients (16.1%). The mortality rate within the whole cohort was 108 patients (13.2%). After controlling for competing risk events (death), the cumulative risk of developing MALE was not statistically different. Prealbumin (hazard ratio [HR], 0.6; 95% confidence interval [CI], 0.41-0.89; P = .010), percutaneous coronary intervention (HR, 2.31; 95% CI, 1.26-4.21; P = .006), Rutherford classification (HR, 1.77; 95% CI, 1.36-2.31; P < .001), white blood cell (HR, 1.85; 95% CI, 1.20-2.87; P = .005), high altitude area (HR, 3.1; 95% CI, 1.43-6.75; P = .004), endovascular treatment (HR, 10.2; 95% CI, 1.44-72.50; P = .020), and length of stay (HR, 1.01; 95% CI, 1.00-1.03; P = .012) were risk factors for MALE. The MALE prediction model had a C-statistic of 0.76 (95% CI, 0.70-0.79). The C-statistic was 0.68 for internal validation and 0.66 for external validation for the MALE prediction model. The MALE prediction model for PAD presented an interactive nomogram and a web-based network calculator. CONCLUSIONS: In this study, the MALE prediction model has a discriminative ability to predict MALE among patients with PAD in frailty. The MALE model can optimize clinical decision-making for patients with PAD in frailty.

OsJAZ4 Fine-Tunes Rice Blast Resistance and Yield Traits.

JAZ proteins function as transcriptional regulators that form a jasmonic acid-isoleucine (JA-Ile) receptor complex with coronatine insensitive 1 (COI1) and regulate plant growth and development. These proteins also act as key mediators in signal transduction pathways that activate the defense-related genes. Herein, the role of OsJAZ4 in rice blast resistance, a severe disease, was examined. The mutation of OsJAZ4 revealed its significance in Magnaporthe oryzae (M. oryzae) resistance and the seed setting rate in rice. In addition, weaker M. oryzae-induced ROS production and expression of the defense genes OsO4g10010, OsWRKY45, OsNAC4, and OsPR3 was observed in osjaz4 compared to Nipponbare (NPB); also, the jasmonic acid (JA) and gibberellin4 (GA4) content was significantly lower in osjaz4 than in NPB. Moreover, osjaz4 exhibited a phenotype featuring a reduced seed setting rate. These observations highlight the involvement of OsJAZ4 in the regulation of JA and GA4 content, playing a positive role in regulating the rice blast resistance and seed setting rate.

Pharmacological profiling of a berbamine derivative for lymphoma treatment.

ABSTRACT: Ca2+/calmodulin-dependent protein kinase II γ (CAMKIIγ) has been identified as a potential target for treating cancer. Based on our previous study of berbamine (BBM) as a CAMKIIγ inhibitor, we have synthesized a new BBM derivative termed PA4. Compared with BBM, PA4 showed improved potency and specificity and was more cytotoxic against lymphoma and leukemia than against other types of cancer. In addition to indirectly targeting c-Myc protein stability, we demonstrated that its cytotoxic effects were also mediated via increased reactive oxygen species production in lymphoma cells. PA4 significantly impeded tumor growth in vivo in a xenograft T-cell lymphoma mouse model. Pharmacokinetics studies demonstrated quick absorption into plasma after oral administration, with a maximum concentration of 1680 ± 479 ng/mL at 5.33 ± 2.31 hours. The calculated oral absolute bioavailability was 34.1%. Toxicity assessment of PA4 showed that the therapeutic window used in our experiments was safe for future development. Given its efficacy, safety, and favorable pharmacokinetic profile, PA4 is a potential lead candidate for treating lymphoma.

Facile Synthesis of ZrO2-SiO2 Antireflective Films with Good Mechanical Performances for Perovskite Solar Cells.

Antireflective (AR) films are widely applied in solar cells to reduce the reflectivity toward sunlight, thus improving the photoelectric conversion efficiency (PCE) of solar cells. However, AR films are still suffering from poor mechanical properties and low transmittance in photovoltaic applications. Herein, a ZrO2-SiO2 composite film with enhanced mechanical properties was successfully synthesized by a facile sol-gel method, whose pencil hardness increased from less than 6B to B compared with the pure SiO2 film synthesized with the same alkali-catalyzed method. Moreover, the ZrO2-SiO2 film with a Zr/Si mole ratio (nZr/Si) of 0.06 exhibited a high transmittance gain (ΔT) of 3.0%, and an obvious increase (1.32%) in PCE was observed in a perovskite solar cell compared with the cell covered by a bare glass. Additionally, both the short-circuit current density (JSC) and PCE of perovskite solar cells have a non-linear increasing relationship with the average transmittance (Tavg) of the ZrO2-SiO2 composite film. In this sense, this work can provide a facile way to prepare AR films effectively improving performances of solar cells.

A mechanical-coupling mechanism in OSCA/TMEM63 channel mechanosensitivity.

Mechanosensitive (MS) ion channels are a ubiquitous type of molecular force sensor sensing forces from the surrounding bilayer. The profound structural diversity in these channels suggests that the molecular mechanisms of force sensing follow unique structural blueprints. Here we determine the structures of plant and mammalian OSCA/TMEM63 proteins, allowing us to identify essential elements for mechanotransduction and propose roles for putative bound lipids in OSCA/TMEM63 mechanosensation. Briefly, the central cavity created by the dimer interface couples each subunit and modulates dimeric OSCA/TMEM63 channel mechanosensitivity through the modulating lipids while the cytosolic side of the pore is gated by a plug lipid that prevents the ion permeation. Our results suggest that the gating mechanism of OSCA/TMEM63 channels may combine structural aspects of the 'lipid-gated' mechanism of MscS and TRAAK channels and the calcium-induced gating mechanism of the TMEM16 family, which may provide insights into the structural rearrangements of TMEM16/TMC superfamilies.

Drug-tolerant persister B-cell precursor acute lymphoblastic leukemia cells.

Reduced responsiveness of precursor B-acute lymphoblastic leukemia (BCP-ALL) to chemotherapy can be inferred when leukemia cells persist after 28 days of initial treatment. Survival of these long-term persister (LTP) / minimal residual disease (MRD) cells is partly due to bone marrow stromal cells that protect them under conditions of chemotherapy stress. We used RNA-seq to analyse BCP-ALL cells that survived a long-term, 30-day vincristine chemotherapy treatment while in co-culture with bone marrow stromal cells. RNAs of as many as 10% of the protein-encoding genes were differentially expressed. There was substantial overlap with genes associated with MRD cell persistence reported in other studies. The top pathway regulated in the LTP cells was that involving p53, a master regulator of a spectrum of responses relevant to drug resistance and cytotoxic drug exposure including control of autophagy. We tested a select number of genes for contribution to BCP-ALL cell survival using Cas9/CRISPR in a 2-step selection, initially for overall effect on cell fitness, followed by 21 days of exposure to vincristine. Many genes involved in autophagy and lysosomal function were found to contribute to survival both at steady-state and during drug treatment. We also identified MYH9, NCSTN and KIAA2013 as specific genes contributing to fitness of BCP-ALL cells. CD44 was not essential for growth under steady state conditions but was needed for survival of vincristine treatment. Finally, although the drug transporter ABCC1/MRP1 is not overexpressed in BCP-ALL, a functional gene was needed for DTP cells to survive treatment with vincristine. This suggests that addition of possible ABCC1 inhibitors during induction therapy could provide benefit in eradication of minimal residual disease in patients treated with a chemotherapy regimen that includes vincristine.

Mechanism underlying delayed rectifying in human voltage-mediated activation Eag2 channel.

The transmembrane voltage gradient is a general physico-chemical cue that regulates diverse biological function through voltage-gated ion channels. How voltage sensing mediates ion flows remains unknown at the molecular level. Here, we report six conformations of the human Eag2 (hEag2) ranging from closed, pre-open, open, and pore dilation but non-conducting states captured by cryo-electron microscopy (cryo-EM). These multiple states illuminate dynamics of the selectivity filter and ion permeation pathway with delayed rectifier properties and Cole-Moore effect at the atomic level. Mechanistically, a short S4-S5 linker is coupled with the constrict sites to mediate voltage transducing in a non-domain-swapped configuration, resulting transitions for constrict sites of F464 and Q472 from gating to open state stabilizing for voltage energy transduction. Meanwhile, an additional potassium ion occupied at positions S6 confers the delayed rectifier property and Cole-Moore effects. These results provide insight into voltage transducing and potassium current across membrane, and shed light on the long-sought Cole-Moore effects.

Improving the Quality of Maternity Care: Learning From Malpractice.

OBJECTIVE: This study aimed to depict the characteristics, injury outcomes, and payment of obstetric malpractice lawsuits to better understand the medicolegal burden in obstetrics and categorize the causes of obstetric malpractice lawsuits using The National Health Service Litigation Authority coding taxonomy for further quality improvement in maternity care. METHODS: We reviewed and retrieved key information on court records of legal trials from China Judgment Online between 2013 and 2021. RESULTS: A total of 3441 obstetric malpractice lawsuits successfully claimed were reviewed in this study, with a total indemnity payment of $139,875,375. After peaking in 2017, the number of obstetric malpractice claims begins to decline. Of the 2424 hospitals that were sued, 8.3% (201/2424) were referred to as "repeat defendant" because they were involved in multiple lawsuits. Death and injury were the outcomes in 53.4% and 46.6% of the cases, respectively. The most common outcome type was neonatal death, which made up 29.8% of all cases. The median indemnity payment for death was higher compared with injury ( P < 0.05). In terms of detailed injury outcomes, the major neonatal injury had higher median indemnity payments than neonatal death and fetal death ( P < 0.05). The median indemnity payment of the major maternal injury was higher than that of maternal death ( P < 0.05). The leading causes of obstetric malpractice were the management of birth complications and adverse events (23.3%), management of labor (14.4%), career decision making (13.7%), fetal surveillance (11.0%), and cesarean section management (9.5%). The cause for 8.7% of cases was high payment (≥$100, 000). As indicated by the results of the multivariate analysis, the hospitals in the midland of China (odds ratio [OR], 0.476; 95% confidence interval [CI], 0.348-0.651), the hospitals in the west of China (OR, 0.523; 95% CI, 0.357-0.767), and the secondary hospitals (OR, 0.587; 95% CI, 0.356-0.967) had lower risks of high payment. Hospitals with ultimate liability (OR, 9.695; 95% CI, 4.072-23.803), full liability (OR, 16.442; 95% CI, 6.231-43.391), major neonatal injury (OR, 12.326; 95% CI, 5.836-26.033), major maternal injury (OR, 20.885; 95% CI, 7.929-55.011), maternal death (OR, 18.783; 95% CI, 8.887-39.697), maternal death with child injury (OR, 54.682; 95% CI, 10.900-274.319), maternal injury with child death (OR, 6.935; 95% CI, 2.773-17.344), and deaths of both mother and child (OR, 12.770; 95% CI, 5.136-31.754) had higher risks of high payment. In the causative domain, only anesthetics had a higher risk of high payment (OR, 5.605; 95% CI, 1.347-23.320), but anesthetic-related lawsuits made up just 1.4% of all cases. CONCLUSIONS: The healthcare systems had to pay a significant amount as a result of obstetric malpractice lawsuits. Greater efforts are required to minimize serious injury outcomes and improve obstetric quality in the risky domains.

RLSegNet: An Medical Image Segmentation Network Based on Reinforcement Learning.

In the area of medical image segmentation, the spatial information can be further used to enhance the image segmentation performance. And the 3D convolution is mainly used to better utilize the spatial information. However, how to better utilize the spatial information in the 2D convolution is still a challenging task. In this paper, we propose an image segmentation network based on reinforcement learning (RLSegNet), which can translate the image segmentation process into a serial of decision-making problem. The proposed RLSegNet is a U-shaped network, which is composed of three components: the feature extraction network, the Mask Prediction Network (MPNet), and the up-sampling network with the cascade attention module. The deep semantic feature in the image is first extracted by adopting the feature extraction network. Then, the Mask Prediction Network (MPNet) is proposed to generate the prediction mask for the current frame based on the prior knowledge (segmentation result). And the proposed cascade attention module is mainly used to generate the weighted feature mask so that the up-sampling network pays more attention to the interesting region. Specifically, the state, action and reward used in the reinforcement learning are redesigned in the proposed RLSegNet to translate the segmentation process as the decision-making process, which performs as the reinforcement learning to realize the brain tumor segmentation. Extensive experiments are conducted on the BRATS 2015 dataset to evaluate the proposed RLSegNet. The experimental results demonstrate that the proposed method can achieve a better segmentation performance, in comparison with other state-of-the-art methods.

SINR- and MI-Based Double-Robust Waveform Design.

Owing to cognitive radar breaking the open-loop receiving-transmitting mode of traditional radar, adaptive waveform design for cognitive radar has become a central issue in radar system research. In this paper, the method of radar transmitted waveform design in the presence of clutter is studied. Since exact characterizations of the target and clutter spectra are uncommon in practice, a single-robust transmitted waveform design method is introduced to solve the problem of the imprecise target spectrum or the imprecise clutter spectrum. Furthermore, considering that radar cannot simultaneously obtain precise target and clutter spectra, a novel double-robust transmitted waveform design method is proposed. In this method, the signal-to-interference-plus-noise ratio and mutual information are used as the objective functions, and the optimization models for the double-robust waveform are established under the transmitted energy constraint. The Lagrange multiplier method was used to solve the optimal double-robust transmitted waveform. The simulation results show that the double-robust transmitted waveform can maximize SINR and MI in the worst case; the performance of SINR and MI will degrade if other transmitted waveforms are employed in the radar system.

Galectin-1 and Galectin-3 in B-Cell Precursor Acute Lymphoblastic Leukemia.

Acute lymphoblastic leukemias arising from the malignant transformation of B-cell precursors (BCP-ALLs) are protected against chemotherapy by both intrinsic factors as well as by interactions with bone marrow stromal cells. Galectin-1 and Galectin-3 are lectins with overlapping specificity for binding polyLacNAc glycans. Both are expressed by bone marrow stromal cells and by hematopoietic cells but show different patterns of expression, with Galectin-3 dynamically regulated by extrinsic factors such as chemotherapy. In a comparison of Galectin-1 x Galectin-3 double null mutant to wild-type murine BCP-ALL cells, we found reduced migration, inhibition of proliferation, and increased sensitivity to drug treatment in the double knockout cells. Plant-derived carbohydrates GM-CT-01 and GR-MD-02 were used to inhibit extracellular Galectin-1/-3 binding to BCP-ALL cells in co-culture with stromal cells. Treatment with these compounds attenuated migration of the BCP-ALL cells to stromal cells and sensitized human BCP-ALL cells to vincristine and the targeted tyrosine kinase inhibitor nilotinib. Because N-glycan sialylation catalyzed by the enzyme ST6Gal1 can regulate Galectin cell-surface binding, we also compared the ability of BCP-ALL wild-type and ST6Gal1 knockdown cells to resist vincristine treatment when they were co-cultured with Galectin-1 or Galectin-3 knockout stromal cells. Consistent with previous results, stromal Galectin-3 was important for maintaining BCP-ALL fitness during chemotherapy exposure. In contrast, stromal Galectin-1 did not significantly contribute to drug resistance, and there was no clear effect of ST6Gal1-catalysed N-glycan sialylation. Taken together, our results indicate a complicated joint contribution of Galectin-1 and Galectin-3 to BCP-ALL survival, with different roles for endogenous and stromal produced Galectins. These data indicate it will be important to efficiently block both extracellular and intracellular Galectin-1 and Galectin-3 with the goal of reducing BCP-ALL persistence in the protective bone marrow niche during chemotherapy.

Targeting disordered-structured domain interactions in Galectin-3 based on NMR and enhanced MD.

Intrinsically disordered regions (IDRs) are common and important functional domains in many proteins. However, IDRs are difficult to target for drug development due to the lack of defined structures that would facilitate the identification of possible drug-binding pockets. Galectin-3 is a carbohydrate-binding protein of which overexpression has been implicated in a wide variety of disorders, including cancer and inflammation. Apart from its carbohydrate-recognition/binding domain (CRD), Galectin-3 also contains a functionally important disordered N-terminal domain (NTD) that contacts the C-terminal domain (CTD) and could be a target for drug development. To overcome challenges involved in inhibitor design due to lack of structure and the highly dynamic nature of the NTD, we used a protocol combining nuclear magnetic resonance data from recombinant Galectin-3 with accelerated molecular dynamics (MD) simulations. This approach identified a pocket in the CTD with which the NTD makes frequent contact. In accordance with this model, mutation of residues L131 and L203 in this pocket caused loss of Galectin-3 agglutination ability, signifying the functional relevance of the cavity. In silico screening was used to design candidate inhibitory peptides targeting the newly discovered cavity, and experimental testing of only three of these yielded one peptide that inhibits the agglutination promoted by wild-type Galectin-3. NMR experiments further confirmed that this peptide indeed binds to a cavity in the CTD, not within the actual CRD. Our results show that it is possible to apply a combination of MD simulations and NMR experiments to precisely predict the binding interface of a disordered domain with a structured domain, and furthermore use this predicted interface for designing inhibitors. This procedure can potentially be extended to many other targets in which similar IDR interactions play a vital functional role.

A miRNA-mediated attenuation of hepatocarcinogenesis in both hepatocytes and Kupffer cells.

MicroRNAs (miRNAs) are small noncoding RNAs that regulate a variety of physiological and pathological functions. miR-26a is one of the many miRNAs that have been identified as regulators of cancer development and as potential anticancer drug targets. However, the specific cellular and molecular mechanisms by which miR-26a attenuates hepatocarcinogenesis are still elusive. Here, we interrogated mouse models with miR-26a cell-specific overexpression in either hepatocytes or myeloid cells to show that miR-26a strongly attenuated the chemical-induced hepatocellular carcinoma (HCC). miR-26a overexpression broadly inhibited the inflammatory response in both hepatocytes and macrophages by decreasing several key oncogenic signaling pathways in HCC promotion. These findings thus reveal new insights into a concerted role of miR-26a in both hepatocytes and Kupffer cells to suppress hepatocarcinogenesis, thereby highlighting the potential use of miR-26a mimetics as potential approaches for the prevention and treatment of HCC.