Tumor-specific activation of folate receptor beta enables reprogramming of immune cells in the tumor microenvironment.

Folate receptors can perform folate transport, cell adhesion, and/or transcription factor functions. The beta isoform of the folate receptor (FRß) has attracted considerable attention as a biomarker for immunosuppressive macrophages and myeloid-derived suppressor cells, however, its role in immunosuppression remains uncharacterized. We demonstrate here that FRß cannot bind folate on healthy tissue macrophages, but does bind folate after macrophage incubation in anti-inflammatory cytokines or cancer cell-conditioned media. We further show that FRß becomes functionally active following macrophage infiltration into solid tumors, and we exploit this tumor-induced activation to target a toll-like receptor 7 agonist specifically to immunosuppressive myeloid cells in solid tumors without altering myeloid cells in healthy tissues. We then use single-cell RNA-seq to characterize the changes in gene expression induced by the targeted repolarization of tumor-associated macrophages and finally show that their repolarization not only changes their own phenotype, but also induces a proinflammatory shift in all other immune cells of the same tumor mass, leading to potent suppression of tumor growth. Because this selective reprogramming of tumor myeloid cells is accompanied by no systemic toxicity, we propose that it should constitute a safe method to reprogram the tumor microenvironment.

A complete pipeline enables haplotyping and phasing macrohaplotype in long sequencing reads for polyploidy samples and a multi-source DNA mixture.

Macrohaplotype combines multiple types of phased DNA variants, increasing forensic discrimination power. High-quality long-sequencing reads, for example, PacBio HiFi reads, provide data to detect macrohaplotypes in multiploidy and DNA mixtures. However, the bioinformatics tools for detecting macrohaplotypes are lacking. In this study, we developed a bioinformatics software, MacroHapCaller, in which targeted loci (i.e., short TRs [STRs], single nucleotide polymorphisms, and insertion and deletions) are genotyped and combined with novel algorithms to call macrohaplotypes from long reads. MacroHapCaller uses physical phasing (i.e., read-backed phasing) to identify macrohaplotypes, and thus it can detect multi-allelic macrohaplotypes for a given sample. MacroHapCaller was validated with data generated from our designed targeted PacBio HiFi sequencing pipeline, which sequenced ∼8-kb amplicon regions harboring 20 core forensic STR loci in human benchmark samples HG002 and HG003. MacroHapCaller also was validated in whole-genome long-read sequencing data. Robust and accurate genotyping and phased macrohaplotypes were obtained with MacroHapCaller compared with the known ground truth. MacroHapCaller achieved a higher or consistent genotyping accuracy and faster speed than existing tools HipSTR and DeepVar. MacroHapCaller enables efficient macrohaplotype analysis from high-throughput sequencing data and supports applications using discriminating macrohaplotypes.

MPKin-YSTR: Interpretation of Y chromosome STR haplotypes for missing persons cases.

Y chromosome Short Tandem Repeat (STR) haplotypes have been used in assisting forensic investigations primarily for identification and male lineage determination. The current SWGDAM interpretation guidelines for Y-STR typing provide helpful guidance on those purposes but do not address the issue of kinship analysis with Y-STR haplotypes. Because of the high mutation rate of Y-STRs, there are complex missing person cases in which inconsistent Y-STR haplotypes between true paternal lineage relatives will arise and cases with two or more male references in the same lineage and yet differ in their haplotypes. Therefore, more useful methods are needed for interpreting the Y-STR haplotype data. Computational methods and interpretation guidelines have been developed specifically addressing this issue, either using a mismatch-based counting method or a pedigree likelihood ratio method. In this study, a software program, MPKin-YSTR, was developed by implementing those more sophisticated methods. This software should be able to improve the interpretation of complex cases with Y-STR haplotype evidence. Thus, more biological evidence will be interpreted, which in turn will result in more investigation leads to help solve crimes.

Biomechanical Comparison of Different Numbers and Configurations of Cross-Links in Long-Segment Spinal Fixation-An Experimental Study in a Porcine Model.

STUDY DESIGN: Biomechanical study. OBJECTIVE: Cross-links are a type of common clinical spinal instrumentation. However, the effects of the position and number of cross-links have never been investigated in long-segment spinal fixation, and the variables have not been optimized. We conducted an in vitro biomechanical study by using a porcine long-segment spinal model with 5 different crosslink configurations to determine the optimal construct for clinical practice. METHODS: Five modalities with paired segmental screws from T15-L5 were tested in 20 porcine spines. The spines without cross-links composed the control group, Group A; those with a single cross-link from L2-3 composed Group B; those with 2 cross-links from L1-2 and L3-4 composed Group C; those with 2 cross-links from T15-L1 and L4-5 composed Group D; and those with 3 cross-links from T15-L1, L2-3 and L4-5 composed Group E. Spinal stiffnesses in flexion, extension, lateral bending, and axial rotation were compared among 5 different cross-link configurations in 5-level porcine spinal units. RESULTS: Flexional, extensional and lateral bending stiffnesses did not significantly change with an increasing number of cross-links or positions in the construct. Axial stiffness was significantly increased with 2 cross-links compared to one (P < 0.05) and with placement more distant from the center of the long spinal fixation construct (P < 0.05). CONCLUSIONS: Two cross-links individually placed proximal and distal from the center of a construct is an optimal and efficient configuration to achieve biomechanical stability in non-rigid lumbar spines undergoing long-level fixation.

Biomechanical comparison of pedicle screw fixation strength among three different screw trajectories using single vertebrae and one-level functional spinal unit.

Three key factors are responsible for the biomechanical performance of pedicle screw fixation: screw mechanical characteristics, bone quality and insertion techniques. To the best of the authors' knowledge, no study has directly compared the biomechanical performance among three trajectories, i.e., the traditional trajectory (TT), modified trajectory (MT) and cortical bone trajectory (CBT), in a porcine model. This study compared the pullout strength and insertion torque of three trajectory methods in single vertebrae, the pullout strength and fixation stiffness including flexion, extension, and lateral bending in a one-level instrumented functional spinal unit (FSU) that mimics the in vivo configuration were clarified. A total of 18 single vertebrae and 18 FSUs were randomly assigned into three screw insertion methods (n = 6 in each trajectory group). In the TT group, the screw converged from its entry point, passed completely inside the pedicle, was parallel to the superior endplate, was located in the superior third of the vertebral body and reached to at least the anterior third of the vertebral body. In the MT group, the convergent angle was similar to that of the TT method but directed caudally to the anterior inferior margin of the vertebral body. The results of insertion torque and pullout strength in single vertebrae were analyzed; in addition, the stiffness and pullout strength in the one-level FSU were also investigated. This study demonstrated that, in single vertebrae, the insertion torque was significantly higher in CBT groups than in TT and MT groups (p < 0.05). The maximal pullout strength was significantly higher in MT groups than in TT and CBT groups (p < 0.05). There was no significant difference in stiffness in the three motions among all groups. The maximal pullout strength in FSUs of MT and CBT groups were significantly higher than the TT groups (p < 0.05). We concluded that either MT or CBT provides better biomechanical performance than TT in single vertebrae or FSUs. The lack of significance of stiffness in FSUs among three methods suggested that MT or CBT could be a reasonable alternative to TT if the traditional trajectory was not feasible.

A machine learning approach for missing persons cases with high genotyping errors.

Estimating the relationships between individuals is one of the fundamental challenges in many fields. In particular, relationship.ip estimation could provide valuable information for missing persons cases. The recently developed investigative genetic genealogy approach uses high-density single nucleotide polymorphisms (SNPs) to determine close and more distant relationships, in which hundreds of thousands to tens of millions of SNPs are generated either by microarray genotyping or whole-genome sequencing. The current studies usually assume the SNP profiles were generated with minimum errors. However, in the missing person cases, the DNA samples can be highly degraded, and the SNP profiles generated from these samples usually contain lots of errors. In this study, a machine learning approach was developed for estimating the relationships with high error SNP profiles. In this approach, a hierarchical classification strategy was employed first to classify the relationships by degree and then the relationship types within each degree separately. As for each classification, feature selection was implemented to gain better performance. Both simulated and real data sets with various genotyping error rates were utilized in evaluating this approach, and the accuracies of this approach were higher than individual measures; namely, this approach was more accurate and robust than the individual measures for SNP profiles with genotyping errors. In addition, the highest accuracy could be obtained by providing the same genotyping error rates in train and test sets, and thus estimating genotyping errors of the SNP profiles is critical to obtaining high accuracy of relationship estimation.

Improved fixation stability for repairing pedicle screw loosening using a modified cement filling technique in porcine vertebrae.

Polymethylmethacrylate (PMMA) has been applied clinically and biomechanically repair loose pedicle screws. Controversies have arisen over data due to uncontrolled cement properties, various locations and sizes of fenestrated holes in repair screws, irregular holes and different bone densities of specimens. In this study, the pullout strength was compared for two techniques, the modified technique to use PMMA to augment a threaded hole and the traditional technique with retrograde injection of a PMMA filling, for standard loose screws in porcine vertebrae. Both techniques provided statistically significant results for sufficiently randomized specimens and experimental procedures. The difference in the pullout strength between conical and cylindrical screws for the aforementioned cement augmentation techniques was also investigated. Twenty-four single-level fresh-frozen lumbar vertebrae from L1 to L6 were harvested from four mature pigs. A total of 0.8 ml of PMMA was retrograde injected into screw holes with a 5.5 mm diameter, followed by insertion of a 5.0 mm diameter repair screw in the traditional group (n = 12). A stiff threaded PMMA hole was created with a 4.5 mm tapping screw before insertion of repair screws in the modified group (n = 12). Two screw geometries were randomly assigned as cylindrical (n = 6) and conical (n = 6) in each group. The correlations between filling techniques, screw geometries and axial pullout strength were analyzed. An appropriate screw trajectory and insertion depth were confirmed using X-ray imaging prior to pullout testing in both groups. For a given screw geometry (cylindrical or conical), the pullout force of the modified group was significantly higher than that of the traditional group. There was no significant difference in the pullout force between the screw geometries for a given filling technique. The cement augmentation technique is far more influential than the screw outer geometry. The modified PMMA technique created a greater anchor force than the traditional method and could be an alternative for revision of pedicle screw loosening.

GP73 is a glucogenic hormone contributing to SARS-CoV-2-induced hyperglycemia.

Severe cases of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are associated with elevated blood glucose levels and metabolic complications. However, the molecular mechanisms for how SARS-CoV-2 infection alters glycometabolic control are incompletely understood. Here, we connect the circulating protein GP73 with enhanced hepatic gluconeogenesis during SARS-CoV-2 infection. We first demonstrate that GP73 secretion is induced in multiple tissues upon fasting and that GP73 stimulates hepatic gluconeogenesis through the cAMP/PKA signaling pathway. We further show that GP73 secretion is increased in cultured cells infected with SARS-CoV-2, after overexpression of SARS-CoV-2 nucleocapsid and spike proteins and in lungs and livers of mice infected with a mouse-adapted SARS-CoV-2 strain. GP73 blockade with an antibody inhibits excessive glucogenesis stimulated by SARS-CoV-2 in vitro and lowers elevated fasting blood glucose levels in infected mice. In patients with COVID-19, plasma GP73 levels are elevated and positively correlate with blood glucose levels. Our data suggest that GP73 is a glucogenic hormone that likely contributes to SARS-CoV-2-induced abnormalities in systemic glucose metabolism.

GP73 is a TBC-domain Rab GTPase-activating protein contributing to the pathogenesis of non-alcoholic fatty liver disease without obesity.

The prevalence of non-obese nonalcoholic fatty liver disease (NAFLD) is increasing worldwide with unclear etiology and pathogenesis. Here, we show GP73, a Golgi protein upregulated in livers from patients with a variety of liver diseases, exhibits Rab GTPase-activating protein (GAP) activity regulating ApoB export. Upon regular-diet feeding, liver-GP73-high mice display non-obese NAFLD phenotype, characterized by reduced body weight, intrahepatic lipid accumulation, and gradual insulin resistance development, none of which can be recapitulated in liver-GAP inactive GP73-high mice. Common and specific gene expression signatures associated with GP73-induced non-obese NAFLD and high-fat diet (HFD)-induced obese NAFLD are revealed. Notably, metformin inactivates the GAP activity of GP73 and alleviates GP73-induced non-obese NAFLD. GP73 is pathologically elevated in NAFLD individuals without obesity, and GP73 blockade improves whole-body metabolism in non-obese NAFLD mouse model. These findings reveal a pathophysiological role of GP73 in triggering non-obese NAFLD and may offer an opportunity for clinical intervention.

Linderae Radix Ethanol Extract Alleviates Diet-Induced Hyperlipidemia by Regulating Bile Acid Metabolism Through gut Microbiota.

Hyperlipidemia is a common metabolic disorder and regarded as one of the main risk factors for cardiovascular disease. The gut microbiota has been identified as a potential contributor to hyperlipidemia as it can greatly regulate bile acid metabolism. Linderae radix is a natural medicine widely used in the treatment of a variety of diseases and is also a common drug for hyperlipidemia. Recently, the lipid-lowering effect of Linderae radix are receiving increasing attention but the underlying mechanism remains unknown. The study aimed to investigate the effects of Linderae radix ethanol extract (LREE) on gut microbiota in rats with hyperlipidemia syndrome. We established a hyperlipidemia rat model using a high-fat diet and used LREE as the intervention. Blood lipid levels and pathological examination were measured to assess the effects of LREE on hyperlipidemia. The gut microbiota was determined by 16s rDNA sequencing and the bile acid metabolism-related proteins were detected by western blot to discover the underlying correlations. The results show that LREE lowered TC, TG, and LDL levels effectively, and it also alleviated liver injury by reducing ALT and AST activity. Meanwhile, LREE improved gut microbiota disturbance caused by HFD via increasing intestinal microbiota diversity and changing the abundance of the Firmicutes, Bacteroidetes, and Actinobacteria. In addition, LREE can increase bile acid reabsorption and promote fecal excretion through farnesoid X receptor (FXR), apical sodium-dependent bile acid transporter (ASBT), organic solute transporter alpha (OST-α), and cytochrome P450 family 7 Subfamily A Member 1 (CYP7A1) thus restoring abnormal bile acid metabolism caused by hyperlipidemia.

Use of longer sized screws is a salvage method for broken pedicles in osteoporotic vertebrae.

Screw loosening due to broken pedicles is a common complication resulting from the insertion of screws either with inadequate diameters or into an osteoporotic pedicle. In this novel in vitro study, we tried to clarify the contribution of the pedicle to screw fixation and subsequent salvage strategies using longer or larger-diameter screws in broken pedicles. Sixty L4 fresh-frozen lumbar vertebrae harvested from mature pigs were designed as the normal-density group (n = 30) and decalcified as the osteoporosis group (n = 30). Three modalities were randomly assigned as intact pedicle (n = 30), semi-pedicle (n = 15), and non-pedicle (n = 15) in each group. Three sizes of polyaxial screws (diameter × length of 6.0 mm × 45 mm, 6.0 mm × 50 mm, and 6.5 mm × 45 mm) over five trials were used in each modality. The associations between bone density, pedicle modality and screw pullout strength were analyzed. After decalcification for 4 weeks, the area bone mineral density decreased to approximately 56% (p < 0.05) of the normal-density group, which was assigned as the osteoporosis group. An appropriate screw trajectory and insertional depth were confirmed using X-ray imaging prior to pullout testing in both groups. The pullout forces of larger-diameter screws (6.5 mm × 45 mm) and longer screws (6.0 mm × 50 mm) were significantly higher (p < 0.05) in the semi- and non-pedicle modalities in the normal-density group, whereas only longer screws (6.0 mm × 50 mm) had a significantly higher (p < 0.05) pullout force in the non-pedicle modalities in the osteoporosis group. The pedicle plays an important role in both the normal bone density group and the osteoporosis group, as revealed by analyzing the pullout force percentage contributed by the pedicle. Use of a longer screw would be a way to salvage a broken pedicle of osteoporotic vertebra.

Biomechanical comparison of pedicle screw fixation strength in synthetic bones: Effects of screw shape, core/thread profile and cement augmentation.

Pedicle screw loosening resulting from insufficient bone-screw interfacial holding power is not uncommon. The screw shape and thread profile are considered important factors of the screw fixation strength. This work investigated the difference in pullout strength between conical and cylindrical screws with three different thread designs. The effects of the thread profiles on the screw fixation strength of cannulated screws with or without cement augmentation in osteoporotic bone were also evaluated. Commercially available artificial standard L4 vertebrae and low-density polyurethane foam blocks were used as substitutes for healthy vertebrae and osteoporotic bones, respectively. The screw pullout strengths of nine screw systems were investigated (six in each). These systems included the combination of three different screw shapes (solid/cylindrical, solid/conical and cannulated/cylindrical) with three different thread profiles (fine-thread, coarse-thread and dual-core/dual-thread). Solid screws were designed for the cementless screw fixation of vertebrae using the standard samples, whereas cannulated screws were designed for the cemented screw fixation of osteoporotic bone using low-density test blocks. Following specimen preparation, a screw pullout test was conducted using a material test machine, and the maximal screw pullout strength was compared among the groups. This study demonstrated that, in healthy vertebrae, both the conical and dual-core/dual-thread designs can improve pullout strength. A combination of the conical and dual-core/dual-thread designs may achieve optimal postoperative screw stability. However, in osteoporotic bone, the thread profile have little impact on the screw fixation strength when pedicle screws are fixed with cement augmentation. Cement augmentation is the most important factor contributing to screw pullout fixation strength as compared to screw designs.

Biomechanical study of the fixation stability of broken pedicle screws and subsequent strategies.

Pedicles are often broken when screws are inserted into hard pedicles with small diameters or when the diameter of the screw itself is inadequate. However, there is a lack of biomechanical literature that addresses screw loosening as a result of broken pedicles or the resulting salvage of those screws. We performed a novel in vitro study to compare the pullout strength of screws between intact pedicles and two different types of broken pedicles; strategies to prevent screw loosening were also compared. Thirty L4 Sawbones were designed to represent intact pedicles, semi-pedicles, and nonpedicles and were prepared for screw insertion. Three sizes of polyaxial screws (diameter × length dimensions of 6.0 mm × 45 mm, 6.0 mm × 50 mm and 6.5 mm × 45 mm) were independently and randomly distributed into the intact-pedicle group (IP group, n = 30), the semi-pedicle group (SP group, n = 15), or the nonpedicle group (NP group, n = 15). The experiments were conducted across a minimum of five trials for each of the chosen screw sizes. We then analyzed the results of the imaging, pullout testing, and embedded bone volume. Any fractures or defects of the vertebrae caused by screw insertion were excluded from the study. The appropriate screw trajectory and insertional depth were confirmed using axial and sagittal X-ray imaging prior to screw pullout testing. A pullout strength of only 41% to 45% for the SP group and 29% to 39% for the NP group was retained following a broken pedicle. The use of longer or larger-diameter screws appears to be an effective salvaging procedure for the NP group (p < 0.05). The embedded bone volume percentage analysis indicated that, compared to the IP group, 68% to 76% of effective bone embedded into the screw threads in the SP group, and 58% to 65% in the NP group. There was no direct correlation between the pullout strength and the embedded bone volume; however, less effective embedded bone volume was associated with lower pullout strength. This study describes the evolution of the well-established screw pullout test being applied to the broken pedicle Sawbone testing model. The pedicle plays an important role in determining the pullout strength of a screw. However, a salvage procedure that utilizes a longer or larger-diameter screw might be a reliable clinical approach to address broken pedicles.

Modification of Mechanical Properties, Polymerization Temperature, and Handling Time of Polymethylmethacrylate Cement for Enhancing Applicability in Vertebroplasty.

Polymethylmethacrylate (PMMA) bone cement is a popular bone void filler for vertebroplasty. However, the use of PMMA has some drawbacks, including the material's excessive stiffness, exothermic polymerization, and short handling time. This study aimed to create an ideal modified bone cement to solve the above-mentioned problems. Modified bone cements were prepared by combining PMMA with three different volume fractions of castor oil (5%, 10%, and 15%). The peak polymerization temperatures, times to achieve the peak polymerization temperature, porosities, densities, modulus and maximum compression strengths of standard (without castor oil), and modified cements were investigated following storage at ambient temperature (22°C) or under precooling conditions (3°C). Six specimens were tested in each group of the aforementioned parameters. Increasing castor oil content and precooling treatment effectively decreased the peak polymerization temperatures and increased the duration to achieve the peak polymerization temperature (P < 0.05). Furthermore, the mechanical properties of the material, including density, modulus, and maximum compression strength, decreased with increasing castor oil content. However, preparation temperature (room temperature versus precooling) had no significant effect (P > 0.05) on these mechanical properties. In conclusion, the addition of castor oil to PMMA followed by precooling created an ideal modified bone cement with a low modulus, low polymerization temperature, and long handling time, enhancing its applicability and safety for vertebroplasty.

A Biomechanical Comparison of Expansive Pedicle Screws for Severe Osteoporosis: The Effects of Screw Design and Cement Augmentation.

Expansive pedicle screws significantly improve fixation strength in osteoporotic spines. However, the previous literature does not adequately address the effects of the number of lengthwise slits and the extent of screw expansion on the strength of the bone/screw interface when expansive screws are used with or without cement augmentation. Herein, four designs for expansive pedicle screws with different numbers of lengthwise slits and different screw expansion levels were evaluated. Synthetic bones simulating severe osteoporosis were used to provide a comparative platform for each screw design. The prepared specimens were then tested for axial pullout failure. Regardless of screw design, screws with cement augmentation demonstrated significantly higher pullout strength than pedicle screws without cement augmentation (p < 0.001). For screws without cement augmentation, solid screws exhibited the lowest pullout strength compared to the four expansive groups (p < 0.01). No significant differences in pullout strength were observed between the expansive screws with different designs (p > 0.05). Taken together, our results show that pedicle screws combined with cement augmentation may greatly increase screw fixation regardless of screws with or without expansion. An increase in both the number of slits and the extent of screw expansion had little impact on the screw-anchoring strength. Cement augmentation is the most influential factor for improving screw pullout strength.

Biomechanical comparison of different combinations of hook and screw in one spine motion unit--an experiment in porcine model.

BACKGROUND: The biomechanical performance of the hooks and screws in spinal posterior instrumentation is not well-characterized. Screw-bone interface failure at the uppermost and lowermost vertebrae is not uncommon. Some have advocated for the use of supplement hooks to prevent screw loosening. However, studies describing methods for combined hook and screw systems that fully address the benefits of these systems are lacking. Thus, the choice of which implant to use in a given case is often based solely on a surgeon's experience instead of on the biomechanical features and advantages of each device. METHODS: We conducted a biomechanical comparison of devices instrumented with different combinations of hooks and screws. Thirty-six fresh low thoracic porcine spines were assigned to three groups (12 per group) according to the configuration used for of fixation: (1) pedicle screw; (2) lamina hook and (3) combination of pedicle screw and lamina hook. Axial pullout tests backward on transverse plane in the direction normal to the rods were performed using a material testing machine and a specially designed grip with self-aligned function. RESULTS: The pullout force for the pedicle screws group was significantly greater than for the hooks and the combination (p < 0.05). However, no significant difference was found between the hooks and the combination (p > 0.05). CONCLUSIONS: Pedicle screws achieve the maximal pullout strength for spinal posterior instrumentation.