Direct Implantation of Patient Brain Tumor Cells into Matching Locations in Mouse Brains for Patient-Derived Orthotopic Xenograft Model Development.

Background: Despite multimodality therapies, the prognosis of patients with malignant brain tumors remains extremely poor. One of the major obstacles that hinders development of effective therapies is the limited availability of clinically relevant and biologically accurate (CRBA) mouse models. Methods: We have developed a freehand surgical technique that allows for rapid and safe injection of fresh human brain tumor specimens directly into the matching locations (cerebrum, cerebellum, or brainstem) in the brains of SCID mice. Results: Using this technique, we successfully developed 188 PDOX models from 408 brain tumor patient samples (both high-and low-grade) with a success rate of 72.3% in high-grade glioma, 64.2% in medulloblastoma, 50% in ATRT, 33.8% in ependymoma, and 11.6% in low-grade gliomas. Detailed characterization confirmed their replication of the histopathological and genetic abnormalities of the original patient tumors. Conclusions: The protocol is easy to follow, without a sterotactic frame, in order to generate large cohorts of tumor-bearing mice to meet the needs of biological studies and preclinical drug testing.

Fractionated radiation therapy alters energy metabolism and induces cellular quiescence exit in patient-derived orthotopic xenograft models of high-grade glioma.

Radiation is one of the standard therapies for pediatric high-grade glioma (pHGG), of which the prognosis remains poor. To gain an in-depth understanding of biological consequences beyond the classic DNA damage, we treated 9 patient-derived orthotopic xenograft (PDOX) models, including one with DNA mismatch repair (MMR) deficiency, with fractionated radiations (2 Gy/day x 5 days). Extension of survival time was noted in 5 PDOX models (P < 0.05) accompanied by γH2AX positivity in >95 % tumor cells in tumor core and >85 % in the invasive foci as well as ∼30 % apoptotic and mitotic catastrophic cell death. The model with DNA MMR (IC-1406HGG) was the most responsive to radiation with a reduction of Ki-67(+) cells. Altered metabolism, including mitochondria number elevation, COX IV activation and reactive oxygen species accumulation, were detected together with the enrichment of CD133+ tumor cells. The latter was caused by the entry of quiescent G0 cells into cell cycle and the activation of self-renewal (SOX2 and BMI1) and epithelial mesenchymal transition (fibronectin) genes. These novel insights about the cellular and molecular mechanisms of fractionated radiation in vivo should support the development of new radio-sensitizing therapies.

TaTPS11 enhances wheat cold resistance by regulating source-sink factor.

The presence of sugar in plant tissue can lead to an increase in the osmotic pressure within cells, a decrease in the freezing point of plants, and protection against ice crystal damage to the tissue. Trehalose is closely related to sucrose, which comprises the largest proportion of sugar and has become a hot topic of research in recent years. Our previous studies have confirmed that a key trehalose synthesis gene, TaTPS11, from the cold-resistant winter wheat DM1, could enhance the cold resistance of plants by increasing sugar content. However, the underlying mechanism behind this phenomenon remains unclear. In this study, we cloned TaTPS11-6D, edited TaTPS11-6D using CRISPR/Cas9 technology and transformed 'Fielder' to obtain T2 generation plants. We screened out OE3-3 and OE8-7 lines with significantly higher cold resistance than that of 'Fielder' and Cri 4-3 edited lines with significantly lower cold resistance than that of 'Fielder'. Low temperature storage limiting factors were measured for OE3-3, OE8-7 and Cri 4-3 treated at different temperatures.The results showed that TaTPS11-6D significantly increased the content of sugar in plants and the transfer of sugar from source to storage organs under cold conditions. The TaTPS11-6D significantly increased the levels of salicylic, jasmonic, and abscisic acids while also significantly decreasing the level of gibberellic acid. Our research improves the model of low temperature storage capacity limiting factor.

Development and validation of a smartwatch algorithm for differentiating physical activity intensity in health monitoring.

To develop and validate a machine learning based algorithm to estimate physical activity (PA) intensity using the smartwatch with the capacity to record PA and determine outdoor state. Two groups of participants, including 24 adults (13 males) and 18 children (9 boys), completed a sequential activity trial. During each trial, participants wore a smartwatch, and energy expenditure was measured using indirect calorimetry as gold standard. The support vector machine algorithm and the least squares regression model were applied for the metabolic equivalent (MET) estimation using raw data derived from the smartwatch. Exercise intensity was categorized based on MET values into sedentary activity (SED), light activity (LPA), moderate activity (MPA), and vigorous activity (VPA). The classification accuracy was evaluated using area under the ROC curve (AUC). The METs estimation accuracy were assessed via the mean absolute error (MAE), the correlation coefficient, Bland-Altman plots, and intraclass correlation (ICC). A total of 24 adults aged 21-34 years and 18 children aged 9-13 years participated in the study, yielding 1790 and 1246 data points for adults and children respectively for model building and validation. For adults, the AUC for classifying SED, MVPA, and VPA were 0.96, 0.88, and 0.86, respectively. The MAE between true METs and estimated METs was 0.75 METs. The correlation coefficient and ICC were 0.87 (p < 0.001) and 0.89, respectively. For children, comparable levels of accuracy were demonstrated, with the AUC for SED, MVPA, and VPA being 0.98, 0.89, and 0.85, respectively. The MAE between true METs and estimated METs was 0.80 METs. The correlation coefficient and ICC were 0.79 (p < 0.001) and 0.84, respectively. The developed model successfully estimated PA intensity with high accuracy in both adults and children. The application of this model enables independent investigation of PA intensity, facilitating research in health monitoring and potentially in areas such as myopia prevention and control.

Patient-Derived Models of Cancer in the NCI PDMC Consortium: Selection, Pitfalls, and Practical Recommendations.

For over a century, early researchers sought to study biological organisms in a laboratory setting, leading to the generation of both in vitro and in vivo model systems. Patient-derived models of cancer (PDMCs) have more recently come to the forefront of preclinical cancer models and are even finding their way into clinical practice as part of functional precision medicine programs. The PDMC Consortium, supported by the Division of Cancer Biology in the National Cancer Institute of the National Institutes of Health, seeks to understand the biological principles that govern the various PDMC behaviors, particularly in response to perturbagens, such as cancer therapeutics. Based on collective experience from the consortium groups, we provide insight regarding PDMCs established both in vitro and in vivo, with a focus on practical matters related to developing and maintaining key cancer models through a series of vignettes. Although every model has the potential to offer valuable insights, the choice of the right model should be guided by the research question. However, recognizing the inherent constraints in each model is crucial. Our objective here is to delineate the strengths and limitations of each model as established by individual vignettes. Further advances in PDMCs and the development of novel model systems will enable us to better understand human biology and improve the study of human pathology in the lab.

Liquid-Metal-Assisted Synthesis of Patterned GaN Thin Films for High-Performance UV Photodetectors Array.

GaN's outstanding physical characteristics allow for a wide range of applications in numerous industries. Although individual GaN-based ultraviolet (UV) photodetectors are the subject of in-depth research in recent decades, the demand for photodetectors array is rising as a result of advances in optoelectronic integration technology. However, as a prerequisite for constructing GaN-based photodetectors array, large-area, patterned synthesis of GaN thin films remains a certain challenge. This work presents a facile technique for pattern growing high-quality GaN thin films for the assembly of an array of high-performance UV photodetectors. This technique uses UV lithography, which is not only very compatible with common semiconductor manufacturing techniques, but also enables precise patterning modification. A typical detector has impressive photo-response performance under 365 nm irradiation, with an extremely low dark current of 40 pA, a high Ilight /Idark ratio over 105 , a high responsivity of 4.23 AW-1 , and a decent specific detectivity of 1.76 × 1012 Jones. Additional optoelectronic studies demonstrate the strong homogeneity and repeatability of the photodetectors array, enabling it to serve as a reliable UV image sensor with enough spatial resolution. These outcomes highlight the proposed patterning technique's enormous potential.

In vitro vascular differentiation system efficiently produces natural killer cells for cancer immunotherapies.

Background: Immunotherapeutic innovation is crucial for limited operability tumors. CAR T-cell therapy displayed reduced efficiency against glioblastoma (GBM), likely due to mutations underlying disease progression. Natural Killer cells (NKs) detect cancer cells despite said mutations - demonstrating increased tumor elimination potential. We developed an NK differentiation system using human pluripotent stem cells (hPSCs). Via this system, genetic modifications targeting cancer treatment challenges can be introduced during pluripotency - enabling unlimited production of modified "off-the-shelf" hPSC-NKs. Methods: hPSCs were differentiated into hematopoietic progenitor cells (HPCs) and NKs using our novel organoid system. These cells were characterized using flow cytometric and bioinformatic analyses. HPC engraftment potential was assessed using NSG mice. NK cytotoxicity was validated using in vitro and in vitro K562 assays and further corroborated on lymphoma, diffuse intrinsic pontine glioma (DIPG), and GBM cell lines in vitro. Results: HPCs demonstrated engraftment in peripheral blood samples, and hPSC-NKs showcased morphology and functionality akin to same donor peripheral blood NKs (PB-NKs). The hPSC-NKs also displayed potential advantages regarding checkpoint inhibitor and metabolic gene expression, and demonstrated in vitro and in vivo cytotoxicity against various cancers. Conclusions: Our organoid system, designed to replicate in vivo cellular organization (including signaling gradients and shear stress conditions), offers a suitable environment for HPC and NK generation. The engraftable nature of HPCs and potent NK cytotoxicity against leukemia, lymphoma, DIPG, and GBM highlight the potential of this innovative system to serve as a valuable tool that will benefit cancer treatment and research - improving patient survival and quality of life.

Effect of electrodialysis on colloidal geometry and dynamics: Why my membrane stack was clogged even after a fine pretreatment?

As a long-standing problem, electrodialysis (ED) clogging is believed a consequence of colloids. However, its blocking causation and clogging mechanism have not been verified. In this study, electrodialysis was used to treat a colloidal saline solution, aiming to answer the question from the "nature" of ED by investigating the influence of ED parameters such as laminar flow, salt concentration, current density and pH on colloid geometry and dynamics during the desalting process. The results revealed that: (i) laminar and membrane electrostatic repulsion and adsorption could not significantly increase the particle size (maximum 2.28 times), while the applied electric field elevated the particle size by 54.52 times (119.9 ± 13.66 to 6537.5 ± 64.35 nm); (ii) when the initial feed concentration elevated 10 times (0.1 to 1 mol/L NaCl), the particle size upsurged 149-fold (5.99 ± 0.57 to >150 µm), and flocs were generated. This enhancement was mainly attributed to the compressive electric double layer effect, and the Debye length was trimmed from 0.96 to 0.30 nm; (iii) The low current density (25 A/m2) had a profound aggregation effect on small BSA particles (roughly 10 nm); (iv) The change of pH causes the conformational transition of BSA. In the strong acidic (pH = 3.0) environment, the colloidal particle size expanded by 13 times. This study confirmed that the aggregation of colloids was the culprit of spacer clogging during electrodialysis at higher salt concentrations (>1 mol/L). Furthermore, experimental data were substituted into the simulation formula to summarise the geometry and dynamic variation of BSA in ED.

Targeting GBM with an Oncolytic Picornavirus SVV-001 alone and in combination with fractionated Radiation in a Novel Panel of Orthotopic PDX models.

BACKGROUND: Animal models representing different molecular subtypes of glioblastoma multiforme (GBM) is desired for developing new therapies. SVV-001 is an oncolytic virus selectively targeting cancer cells. It's capacity of passing through the blood brain barrier makes is an attractive novel approach for GBM. MATERIALS AND METHODS: 23 patient tumor samples were implanted into the brains of NOD/SCID mice (1 × 105 cells/mouse). Tumor histology, gene expression (RNAseq), and growth rate of the developed patient-derived orthotopic xenograft (PDOX) models were compared with the originating patient tumors during serial subtransplantations. Anti-tumor activities of SVV-001 were examined in vivo; and therapeutic efficacy validated in vivo via single i.v. injection (1 × 1011 viral particle) with or without fractionated (2 Gy/day x 5 days) radiation followed by analysis of animal survival times, viral infection, and DNA damage. RESULTS: PDOX formation was confirmed in 17/23 (73.9%) GBMs while maintaining key histopathological features and diffuse invasion of the patient tumors. Using differentially expressed genes, we subclassified PDOX models into proneural, classic and mesenchymal groups. Animal survival times were inversely correlated with the implanted tumor cells. SVV-001 was active in vitro by killing primary monolayer culture (4/13 models), 3D neurospheres (7/13 models) and glioma stem cells. In 2/2 models, SVV-001 infected PDOX cells in vivo without harming normal brain cells and significantly prolonged survival times in 2/2 models. When combined with radiation, SVV-001 enhanced DNA damages and further prolonged animal survival times. CONCLUSION: A panel of 17 clinically relevant and molecularly annotated PDOX modes of GBM is developed, and SVV-001 exhibited strong anti-tumor activities in vitro and in vivo.

Resource recovery from RO concentrate using nanofiltration: Impact of active layer thickness on performance.

Modelling the removal of monovalent and divalent ions from seawater via nanofiltration is crucial for pre-treatment in seawater reverse osmosis systems. Effective separation of divalent ions through nanofiltration and allowing the permeate containing only monovalent ions to pass through the reverse osmosis system produces pure NaCl salt from the concentrate. However, the Donnan steric pore model and dielectric exclusion assume a uniformly distributed cylinder pore morphology, which is not representative of the actual membrane structure. This study analyzed the impact of membrane thickness on neutral solute removal and investigated the effect of two different methods for calculating the Peclet number on rejection rates of monovalent and divalent salts. Results show that membrane thickness has a significant effect on rejection rates, particularly for uncharged solutes in the range of 0.5-0.7 solute radius to membrane pore size ratio. Operating pressures above 10 bar favour the use of effective active layer thickness over the membrane pore size to calculate the Peclet number. At low pressures, using the effective active layer can lead to overestimation of monovalent salt rejection and underestimation of divalent salt rejection. This study highlights the importance of appropriate Peclet number calculation methods based on applied pressure when modelling membrane separation performance.

How great is the stabilization of crowded polyphenylbiphenyls by London dispersion?

Decaphenylbiphenyl (1) and 2,2',4,4',6,6'-hexaphenylbiphenyl (2) are bulky molecules expected to be greatly destabilized by steric crowding. Herein, through a combined experimental and computational approach, we evaluate the molecular energetics of crowded biphenyls. This is complemented by the study of phase equilibria for 1 and 2. Compound 1 shows a rich phase behavior, displaying an unusual interconversion between two polymorphs. Surprisingly, the polymorph with distorted molecules of C1 symmetry is found to have the highest melting point and to be the one that is preferentially formed. The thermodynamic results also indicate that the polymorph displaying the more regular D2 molecular geometry has larger heat capacity and is probably the more stable at lower temperatures. The melting and sublimation data clearly reveal the weakening of cohesive forces in crowded biphenyls due to the lower molecular surface area. The experimental quantification of the intramolecular interactions in 1 and 2 indicated, using homodesmotic reactions, a molecular stabilization of about 30 kJ mol-1. We attribute the origin of this stabilization in both compounds to the existence of two parallel-displaced π⋯π interactions between the ortho-phenyl substituents on each side of the central biphenyl. Computational calculations with dispersion-corrected DFT methods underestimate the stabilization in 1, unless the steric crowding is well balanced in a homodesmotic scheme. This work demonstrates that London dispersion forces are important in crowded aromatic systems, making these molecules considerably more stable than previously thought.

Automatic Segmentation of Hyperreflective Foci in OCT Images Based on Lightweight DBR Network.

Hyperreflective foci (HF) reflects inflammatory responses for fundus diseases such as diabetic macular edema (DME), retina vein occlusion (RVO), and central serous chorioretinopathy (CSC). Shown as high contrast and reflectivity in optical coherence tomography (OCT) images, automatic segmentation of HF in OCT images is helpful for the prognosis of fundus diseases. Previous traditional methods were time-consuming and required high computing power. Hence, we proposed a lightweight network to segment HF (with a speed of 57 ms per OCT image, at least 150 ms faster than other methods). Our framework consists of two stages: an NLM filter and patch-based split to preprocess images and a lightweight DBR neural network to segment HF automatically. Experimental results from 3000 OCT images of 300 patients (100 DME,100 RVO, and 100 CSC) revealed that our method achieved HF segmentation successfully. The DBR network had the area under curves dice similarity coefficient (DSC) of 83.65%, 76.43%, and 82.20% in segmenting HF in DME, RVO, and CSC on the test cohort respectively. Our DBR network achieves at least 5% higher DSC than previous methods. HF in DME was more easily segmented compared with the other two types. In addition, our DBR network is universally applicable to clinical practice with the ability to segment HF in a wide range of fundus diseases.

Individualized Statistical Modeling of Lesions in Fundus Images for Anomaly Detection.

Anomaly detection in fundus images remains challenging due to the fact that fundus images often contain diverse types of lesions with various properties in locations, sizes, shapes, and colors. Current methods achieve anomaly detection mainly through reconstructing or separating the fundus image background from a fundus image under the guidance of a set of normal fundus images. The reconstruction methods, however, ignore the constraint from lesions. The separation methods primarily model the diverse lesions with pixel-based independent and identical distributed (i.i.d.) properties, neglecting the individualized variations of different types of lesions and their structural properties. And hence, these methods may have difficulty to well distinguish lesions from fundus image backgrounds especially with the normal personalized variations (NPV). To address these challenges, we propose a patch-based non-i.i.d. mixture of Gaussian (MoG) to model diverse lesions for adapting to their statistical distribution variations in different fundus images and their patch-like structural properties. Further, we particularly introduce the weighted Schatten p-norm as the metric of low-rank decomposition for enhancing the accuracy of the learned fundus image backgrounds and reducing false-positives caused by NPV. With the individualized modeling of the diverse lesions and the background learning, fundus image backgrounds and NPV are finely learned and subsequently distinguished from diverse lesions, to ultimately improve the anomaly detection. The proposed method is evaluated on two real-world databases and one artificial database, outperforming the state-of-the-art methods.

Epigenetic Alterations of Repeated Relapses in Patient-matched Childhood Ependymomas.

Recurrence is frequent in pediatric ependymoma (EPN). Our longitudinal integrated analysis of 30 patient-matched repeated relapses (3.67 ± 1.76 times) over 13 years (5.8 ± 3.8) reveals stable molecular subtypes (RELA and PFA) and convergent DNA methylation reprogramming during serial relapses accompanied by increased orthotopic patient derived xenograft (PDX) (13/27) formation in the late recurrences. A set of differentially methylated CpGs (DMCs) and DNA methylation regions (DMRs) are found to persist in primary and relapse tumors (potential driver DMCs) and are acquired exclusively in the relapses (potential booster DMCs). Integrating with RNAseq reveals differentially expressed genes regulated by potential driver DMRs (CACNA1H, SLC12A7, RARA in RELA and HSPB8, GMPR, ITGB4 in PFA) and potential booster DMRs (PLEKHG1 in RELA and NOTCH, EPHA2, SUFU, FOXJ1 in PFA tumors). DMCs predicators of relapse are also identified in the primary tumors. This study provides a high-resolution epigenetic roadmap of serial EPN relapses and 13 orthotopic PDX models to facilitate biological and preclinical studies.

Liquid-metal based flexible a-IZTO ultrathin films for electrical and optical applications.

Amorphous indium zinc tin oxide (a-IZTO) is a kind of transparent conductive oxide (TCO), which can be used in transparent electrodes, transistors, and flexible devices. At present, a key limitation of a-IZTO is the costly vacuum manufacturing technology, and its commercial production is also restricted by the complex raw material preparation process. In this article, we report a liquid metal-based van der Waals (vdW) exfoliation technique by which a-IZTO films with several nanometres thickness are fabricated. The a-IZTO films fabricated in ambient air have a size on the centimeter scale and an optical transmittance of 99.64%; they are also large-area flexible oxide films. In order to illustrate the capabilities of this technology, we fabricated thin film transistors (TFTs) and photodetectors based on a-IZTO films. An a-IZTO thin film transistor (TFT) has an on/off ratio of 106. When the Vds is 5 V, the responsivity, detectivity and external quantum efficiency of an a-IZTO photodetector are 7.57 × 104 A W-1, 4.00 × 1015 Jones and 3.68 × 105%, respectively, exhibiting one of the top performances in this field.

Type-I Heterostructure Based on WS2/PtS2 for High-Performance Photodetectors.

van der Waals (vdW) heterodiodes composed of two-dimensional (2D) layered materials led to a new prospect in photoelectron diodes and photovoltaic devices. Existing studies have shown that Type-I heterostructures have great potential to be used as photodetectors; however, the tunneling phenomena in Type-I heterostructures have not been fully revealed. Herein, a highly efficient nn+ WS2/PtS2 Type-I vdW heterostructure photodiode is constructed. The device shows an ultrahigh reverse rectification ratio of 105 owing to the transmission barrier-induced low reverse current. A unilateral depletion region is formed on WS2, which inhibits the recombination of carriers at the interface and makes the external quantum efficiency (EQE) of the device reach 67%. Due to the tunneling mechanism of the device, which allows the co-existence of a large photocurrent and a low dark current, this device achieves a light on/off ratio of over 105. In addition, this band design allows the device to maintain a high detectivity of 4.53 × 1010 Jones. Our work provides some new ideas for exploring new high-efficiency photodiodes.

Anomaly detection in fundus images by self-adaptive decomposition via local and color based sparse coding.

Anomaly detection in color fundus images is challenging due to the diversity of anomalies. The current studies detect anomalies from fundus images by learning their background images, however, ignoring the affluent characteristics of anomalies. In this paper, we propose a simultaneous modeling strategy in both sequential sparsity and local and color saliency property of anomalies are utilized for the multi-perspective anomaly modeling. In the meanwhile, the Schatten p-norm based metric is employed to better learn the heterogeneous background images, from where the anomalies are better discerned. Experiments and comparisons demonstrate the outperforming and effectiveness of the proposed method.

The Pursuit of Perphenylterphenyls.

Tetradecaphenyl-p-terphenyl (2) was synthesized from 2,3,5,6-tetraphenyl-1,4-diiodobenzene (11) by two methods. Ullmann coupling of 11 with pentaphenyliodobenzene (9) gave compound 2 in 1.7 % yield, and Sonogashira coupling of 11 with phenylacetylene, followed by a double Diels-Alder reaction of the product diyne 12 with tetracyclone (6), gave 2 in 1.5 % overall yield. The latter reaction also gave the monoaddition product 4-(phenylethynyl)-2,2',3,3',4',5,5',6,6'-nonaphenylbiphenyl (13) in 4 % overall yield. The X-ray structures of compounds 2 and 13 show them to possess core aromatic rings distorted into shallow boat conformations. Density functional calculations indicate that these unusual structures are not the lowest energy conformations in the gas phase and may be the result of packing forces in the crystal. In addition, while uncorrected DFT calculations indicate that the strain energy in compound 2 is approximately 50 kcal/mol, dispersion-corrected DFT calculations suggest that it is essentially unstrained, due to compensating, favorable, intramolecular interactions of its many phenyl rings. An attempted synthesis of tetradecaphenyl-o-terphenyl (4) by reaction of diphenylhexatriyne (14) with three equivalents of tetracyclone at 350 °C gave only the diadduct 2-(phenylethynyl)-2',3,3',4,4',5,5',6,6'-nonaphenylbiphenyl (15) in 17 % yield. Even higher temperatures failed to produce 4 and lowered the yield of 15, perhaps due to rapid decomposition of the starting materials. Ullmann coupling of 3,4,5,6-tetraphenyl-1,2-diiodobenzene (16) and compound 9 also failed to give compound 4.

Association between Different Grades of Myopic Tractional Maculopathy and OCT-Based Macular Scleral Deformation.

Purpose: To investigate the characteristics of macular outward scleral height (MOSH) in different grades of myopic tractional maculopathy (MTM) and explore the risk factors for MTM. Methods: A total of 188 eyes (188 participants) with high myopia were divided into the no MTM (nMTM) group and the MTM group, which was further graded into foveoschisis, foveal detachment, full-thickness macular hole, and macular hole with retinal detachment. Swept-source optical coherence tomography was used to measure the MOSH. Results: No significant differences were found in axial length between the nMTM and MTM groups (p = 0.295). The MOSH was significantly higher in the MTM group (p < 0.001), which was identified as a risk factor for MTM (OR = 1.108, p < 0.001). The proportion of eyes with severe atrophic myopic maculopathy (AMM) was higher in the MTM group (28.48%) (p = 0.003). The macular hole with foveoschisis (MH/FS+) subgroup presented a higher average MOSH (p = 0.012) and more severe AMM (p = 0.009) than the macular hole without foveoschisis (MH/FS−) subgroup. Conclusion: MOSH would be more suitable for estimating MTM occurrence than axial length. The grading of AMM helps to evaluate the severity of MTM. The categorization of MH/FS− as a distinct grade from MH/FS+ might be preferable.