Normal Numbers Research Articles

Enhancing citrus fruit yield investigations through flight height optimization with UAV imaging

Citrus fruit yield is essential for market stability, as it allows businesses to plan for production and distribution. However, yield estimation is a complex and time-consuming process that often requires a large number of field samples to ensure representativeness. To address this challenge, we investigated the optimal altitude for unmanned aerial vehicle (UAV) imaging to estimate the yield of Citrus unshiu fruit. We captured images from five different altitudes (30 m, 50 m, 70 m, 90 m, and 110 m), and determined that a resolution of approximately 5 pixels/cm is necessary for reliable estimation of fruit size based on the average diameter of C. unshiu fruit (46.7 mm). Additionally, we found that histogram equalization of the images improved fruit count estimation compared to using untreated images. At the images from 30 m height, the normal image estimates fruit numbers as 73, 55, and 88. However, the histogram equalized image estimates 88, 71, 105. The actual number of fruits is 124, 88, and 141. Using a Vegetation Index such as IPCA showed a similar estimation value to histogram equalization, but I1 estimation represents a gap to actual yields. Our results provide a valuable database for future UAV field investigations of citrus fruit yield. Using flying platforms like UAVs can provide a step towards adopting this sort of model spanning ever greater regions at a cheap cost, with this system generating accurate results in this manner.

Macro-micro numerical analysis of granular materials considering principal stress rotation based on DEM simulation of dynamic hollow cylinder test

The granular materials were subjected to the principal stress rotation effect induced by traffic loads, wave loads, and earthquake loads in geotechnical engineering practice. In order to reveal the influence of principal stress rotation on the macro-micro dynamic characteristics, this study proposed a numerical simulation method modeling physical dynamic hollow cylinder tests. Taking the dynamic stress state induced by traffic loading as an example, the numerical test model of the dynamic hollow cylinder was conducted on the subgrade soil, and the influence of principal stress rotation on the macro-micro evolution of dynamic characteristics was analyzed.The results showed that the principal stress rotation accelerated the development of vertical accumulative plastic strain and particle displacement, which aggravated micro-structure destruction of granular material. Moreover, the increase of confining stress declined the variations of normal contact numbers in each direction, which resisted the shear stress induced by principal stress rotation. This study provided a novel method to investigate the macro-micro dynamic characteristics of granular materials subjected to principal stress rotation, which made up for the inability of physical hollow cylinder tests to study microscopic evolution.

The ion channel TRPV5 regulates B-cell signaling and activation.

B-cell activation triggers the release of endoplasmic reticulum calcium stores through the store-operated calcium entry (SOCE) pathway resulting in calcium influx by calcium release-activated calcium (CRAC) channels on the plasma membrane. B-cell-specific murine knockouts of SOCE do not impact humoral immunity suggesting that alternative channels may be important. We identified a member of the calcium-permeable transient receptor potential (TRP) ion channel family, TRPV5, as a candidate channel expressed in B cells by a quantitative polymerase chain reaction (qPCR) screen. To further investigate the role of TRPV5 in B-cell responses, we generated a murine TRPV5 knockout (KO) by CRISPR-Cas9. We found TRPV5 polarized to B-cell receptor (BCR) clusters upon stimulation in a PI3K-RhoA-dependent manner. TRPV5 KO mice have normal B-cell development and mature B-cell numbers. Surprisingly, calcium influx upon BCR stimulation in primary TRPV5 KO B cells was not impaired; however, differential expression of other calcium-regulating proteins, such as ORAI1, may contribute to a compensatory mechanism for calcium signaling in these cells. We demonstrate that TRPV5 KO B cells have impaired spreading and contraction in response to membrane-bound antigen. Consistent with this, TRPV5 KO B cells have reduced BCR signaling measured through phospho-tyrosine residues. Lastly, we also found that TRPV5 is important for early T-dependent antigen specific responses post-immunization. Thus, our findings identify a role for TRPV5 in BCR signaling and B-cell activation.

Mast cells, mediators, and symptomatic activation

<abstract> <p>Mast cells (MC) are central effectors of allergic disease and distinct subsets with varying amounts of tryptase, chymase, and carboxypeptidase A3, and cathepsin G is distributed throughout the body. Their involvement in a diverse range of non-allergic illnesses mediated by a complex range of preformed and newly synthesized mediators is now increasingly recognized. The latter especially include conditions under the umbrella term of mast cell activation syndrome. In allergic disease, much has been written about the mechanisms by which the early and rapidly acting mediators produce both localized and systemic allergic symptoms. The role of chymase is presently underappreciated but there is increased awareness that MCs contain significant amounts of preformed TNF alpha and synthesize and releases a wide range of inflammatory cytokines such as interleukins (IL) 1β, IL6, IL31, and IL33. These can aggravate itching and perpetuate inflammation and likely contribute to the late constitutional symptoms seen in allergic reactions. Importantly, their involvement helps to clarify the role of MCs in stress and non-parasitic infections. Presently, unexplained is the increasing incidence of significant acute allergic reactions within a relatively short time frame. In this context, there is increasing interest in the environmental, menstrual, endocrine, circadian, and psychological factors that influence MC activation as well as the endocrine pathways involving the renin angiotensin system that oppose hypotension. In non-allergic diseases with normal numbers of MCs, reduced thresholds for activation may be produced by various combinations of life and dietary factors. Diagnosing these conditions is difficult but may be helped by urinary analysis of prostaglandin metabolites. The investigation and management of mastocytosis with and without mutations of c-kit is also relevant to allergic disease and the new medications used may also be helpful in idiopathic anaphylaxis. This knowledge may open a new chapter in human diseases and mast cell regulation.</p> </abstract>

Adansonia digitata L. Stem Bark Attenuates Epileptic Seizure, Depression, and Neurodegeneration by Mediating GABA and Glutamate in Pentylenetetrazol-Kindled Rats.

Epilepsy is a neurological condition characterized by repeated seizures attributable to synchronous neuronal activity in the brain. The study evaluated the effect of acetone extract of Adansonia digitata stem bark (ASBE) on seizure score, cognition, depression, and neurodegeneration as well as the level of Gamma-Aminobutyrate acid (GABA) and glutamate in Pentylenetetrazol-kindled rats. Thirty-five rats were assigned into five groups (n = 7). Groups 1-2 received normal saline and 35 mg/kg PTZ every other day. Groups 3-4 received 125 mg/kg and 250 mg/kg ASBE orally while group 5 received 5 mg/kg diazepam daily for twenty-six days. Group 3-5 received PTZ every other day, 30 mins after ASBE and diazepam. The results showed that Pentylenetetrazol (PTZ) induces seizure, reduces mobility time in force swim test and decreases the normal cell number in the brain. It also significantly decreases (p < 0.05) catalase, superoxide dismutase and reduced glutathione activities compared to the ASBE pre-treated rats. Pre-treatment with ASBE reportedly decreases seizure activities significantly (p < 0.05) and increases mobility time in the force swim test. ASBE also significantly elevate (p < 0.05) the normal cell number in the hippocampus, temporal lobe, and dentate gyrus. ASBE reduced seizure activity and prevented depression in PTZ-treated rats. It also prevented neurodegeneration by regulating glutamate and GABA levels in the brain as well as preventing lipid peroxidation.

Refractory alopecia areata with single hairs imitating frontal fibrosing alopecia: a prospective observational study.

Lonely hair sign is considered as a clue to the diagnosis of frontal fibrosing alopecia (FFA). To report an undescribed variant of alopecia areata (AA) with which the patient developed single hairs and other features similar to FFA and to determine the underlying mechanism. We conducted a prospective observational study in patients who presented with receding hairline and single hairs, evaluating the clinical, trichoscopic, and histological features and their correlation. Immunochemistry studies were performed to describe the microenvironment. Eighteen patients were enrolled in the study. Despite the similarity to FFA clinically, these patients showed different histopathology which revealed a normal number of pilosebaceous units, one anagen hair in one or more pilosebaceous units, and others in telogen stage, consistent with single hairs under the naked eye or under trichoscopy. The severity of the hair loss assessed by SALT was no more than 50, but the response to conventional therapy was poor. This study reports a unique variant of AA. The pathological basis is an increase in the telogen hair follicles, with one anagen hair in one or more pilosebaceous units. Minimal inflammation consisting of CD3+ T lymphocytes and mast cells was demonstrated in the microenvironment.

Microwave assistance effect for rock breaking of TBM disc cutter using the coupled method of continuum and grain-based model

Microwave-assisted rock breaking technique can significantly weaken the strength and the stiffness of hard rock, and can consequently enhance TBM efficiency as well as relieve wear of disc cutters. Understanding the mechanism of microwave-assisted rock breaking requires knowledge of the intersection and connectivity properties of freshly formed fractures below the cutter and the fracture network resulting from microwave-induced damage. In this study, a numerical approach, coupled with the grain-based model (GBM) and continuum based microwave electromagnetic analysis, for simulating the microwave-treated granite is proposed firstly. Subsequently, the numerical experiment of TBM cutter penetration is conducted with the consideration of five levels of microwave power irradiation as well as two waveguide arrangements. The temperature field and fracture behavior are analyzed to investigate the thermal damage of the granite after the treatment of the microwave. Furthermore, the reduction of peak normal force and variation of average coordination number are examined to evaluate the impact of waveguide position on the effectiveness of microwave-assisted rock breaking. The simulation results indicate that the suitable power of microwave irradiation (≥ 30 kW) can promote the rise of granite temperature directly below the waveguide, further resulting in an increase in the quantity of microcracks and noticeable damage to the rock block. When the microwave power exceeds 30 kW, the peak normal force and average coordination number progressively start to decline for the case of a waveguide port facing the centerline of a single cutter. In the case of waveguide facing the gap between double cutters, the decreasing trend of the peak normal force is similar to that under the condition of single cutter, but the starting threshold has been raised to 45 kW. When the microwave power is below 60 kW, the average coordination does not change significantly. After comparing the improvement effects of microwave-assisted rock breaking under the two waveguide arrangements, a preferred condition of the microwave for breaking is determined. The waveguide port facing the centerline of a single cutter, and the microwave power of 30 ∼ 60 kW can lead a success in a satisfactory effect in term of the reduction rate of peak normal force and average coordination number.

Non-Axisymmetric Bouncing Dynamics on a Moving Superhydrophobic Surface

The phenomenon of droplet impact on moving surfaces is widely observed in fields such as transportation, rotating machinery, and inkjet printing. Droplets exhibit non-axisymmetric behavior due to the motion of solid surfaces which significantly determines core parameters such as contact time, maximum spreading radius, and bounding velocity, thereby affecting the efficiency of related applications. In this study, we focus on the kinetics and morphology of the non-axisymmetric bouncing behaviors for droplets impacting on a moving superhydrophobic surface (SHPS) within the normal (Wen) and tangential (Wet) Weber numbers. Considering the influences of the moving surface on the contact area and contact time, the previous scaling formula for the horizontal velocity of droplets has been improved. Based on the velocity superposition hypothesis, we establish a theoretical model for the ratio of the maximum spreading radius at both ends depending on Wen and Wet. This research provides both experimental and theoretical evidence for understanding and controlling the non-axisymmetric behavior of droplets impacting on moving surfaces.

Uncertain linear programming with cloud set constraints integrating fuzziness and randomness

In reality, there is a significant need for processing methods of uncertain programming problems with fuzziness and randomness. Although there are many extensions of fuzzy sets to handle uncertain information, it is rare to organically integrate randomness and fuzziness, such as the cloud set which is the set extension of cloud model. To address the limitations of uncertain linear programming, which lacks consideration of fuzziness and randomness, a new approach called uncertain linear programming with cloud set constraints is proposed. This approach integrates both fuzziness and randomness properties. Firstly, the definition of uncertain linear programming model with cloud set constraints integrating fuzzy and randomness properties is given. Secondly, the construction method of cloud set membership function of uncertain linear programming model is studied. Thirdly, the solution method for uncertain linear programming model based on Ioperation and P operation in cloud set is argued. Finally, the effectiveness of the method is verified through a specific application example and a comparison case with other linear programs. This method of uncertain linear programming with cloud set constraints utilizes the normal cloud set to represent the objective and conditional constraints, effectively capturing the fuzziness and randomness inherent in these constraints. The contribution of this study is that it proposes the use of random membership degree to replace the subjective fixed single membership in the constraints of linear programming. The membership degree of normal cloud set is expressed by a normal random number with a stable tendency, which integrates fuzziness and randomness with a regular, discrete, sparse thickness instead of a fixed curve. It can more effectively express and adapt to the inherent uncertainty of the objective world because various variables in natural science and social science typically follow a normal distribution. So, it has good objectivity and universality.

A role for mutations in AK9 and other genes affecting ependymal cells in idiopathic normal pressure hydrocephalus.

Idiopathic normal pressure hydrocephalus (iNPH) is an enigmatic neurological disorder that develops after age 60 and is characterized by gait difficulty, dementia, and incontinence. Recently, we reported that heterozygous CWH43 deletions may cause iNPH. Here, we identify mutations affecting nine additional genes (AK9, RXFP2, PRKD1, HAVCR1, OTOG, MYO7A, NOTCH1, SPG11, and MYH13) that are statistically enriched among iNPH patients. The encoded proteins are all highly expressed in choroid plexus and ependymal cells, and most have been associated with cilia. Damaging mutations in AK9, which encodes an adenylate kinase, were detected in 9.6% of iNPH patients. Mice homozygous for an iNPH-associated AK9 mutation displayed normal cilia structure and number, but decreased cilia motility and beat frequency, communicating hydrocephalus, and balance impairment. AK9+/- mice displayed normal brain development and behavior until early adulthood, but subsequently developed communicating hydrocephalus. Together, our findings suggest that heterozygous mutations that impair ventricular epithelial function may contribute to iNPH.

Early Generation Telomerase Deficient Mice to Investigate Clonal Hematopoiesis in Aging

Clonal hematopoiesis is an age-associated condition characterized by the expansion of hematopoietic stem cell (HSC) pools with somatic mutations, observed in 10-30% of individuals older than 70 years. This phenomenon is primarily driven by mutations in transcriptional regulators such as DNMT3A, TET2, and ASXL1. While the clonal expansion of mutated HSCs is ubiquitous with age, it appears largely benign in the absence of strong selective pressures. These benign clones might compensate for the loss of regenerative HSC capacity during aging, and skew differentiation towards the myeloid lineage. Telomere erosion plays a significant role in this process. Telomeres normally prevent the activation of the DNA damage response (DDR) machinery. However, with each cell division, telomeres shorten until they reach a critical length, eventually leading to apoptosis or senescence. Previous studies have suggested a potential involvement of telomere attrition in the development of clonal hematopoiesis (Hinds et al. Blood 2016; Zink et al. Blood 2017). Late generation telomerase deficient (Terc-/- G5) mice develop premature bone marrow failure and mortality, however earlier generation Terc-/- G3 (3 homozygous crosses) have shortened telomeres but appear relatively normal. Importantly, inhibitors of telomerase have recently entered clinical trials for haematological malignancies and therefore understanding the long-term effects of telomerase inhibition is important. By studying the interplay between telomere attrition, HSC aging, and the development of CHIP, we aim to gain insights into the mechanisms underlying age-associated hematopoietic changes and their potential clinical implications. We examined young (age 8-10 week) and aged (10-12 month) Terc-/- G3 mice. Telomere lengths were significantly reduced in bone marrow cells from young Terc G3 (15 kb) when compared to wild-type mice (30 kb) with mild reduction in BM cellularity. Absolute long-term HSC numbers were reduced, as were megakaryocyte erythroid progenitors, but there was preservation of granulocyte macrophage progenitors and common myeloid progenitors. Spleen progenitor numbers were not affected. In comparison, aged (10-12 months old) Terc G3 mice showed enhanced differentiation skewing in the peripheral blood with significantly increased myeloid (n = 8 wt; n = 12 Terc ko; p = 0.0004) and concomitantly reduced B cell (p = 0.0002) and T cell (p = 0.0320) populations. There was marked mobilization of bone marrow progenitors to the spleen, most notably GMP (p = 0.0004) but also MEP and CMP in aged Terc G3. Furthermore, aged Terc G3 ko now exhibited marked reduction in bone marrow CMP (p &amp;lt; 0.0001) and MEP (p &amp;lt; 0.0001), but skewing towards GMP (p&amp;lt;0.001) resulting in normal total GMP numbers, but markedly reduced CMP and MEP numbers (p &amp;lt; 0.0001). Compared to young Terc G3 mice, the aged Terc G3 mice had relative expansion of the BM LT-HSC population, and therefore normal numbers of BM LT-HSC compared to aged matched controls. Aged Terc G3 BM LTHSC have been isolated for gene expression and clonality analysis. Together, these findings are consistent with accelerated aging in G3 Terc-/- mice, providing a model for examining clonal hematopoiesis. In order to assess the cell intrinsic function of Terc G3 LTHSC, we next performed non-competitive bone marrow transplantation experiments from Terc ko or wild-type donors into lethally irradiated young wild-type recipients (n = 9 per group), and analysed these at 4 months post-transplant. There was reduced, but stable reconstitution by Terc G3 cells, consistent with previous competitive bone marrow transplants in Terc G3 (Bruedigam et al. Cell Stem Cell 2014). We observed a similar reduction in bone marrow cellularity in Terc ko transplanted recipients (p = 0.003). Lineage skewing was less pronounced but CMP percentages were significantly reduced in Terc ko compared to wild-type conditions (p = 0.03). In contrast to primary mice, mobilization of progenitors to the spleen was not observed, suggesting that this finding was ameliorated by normal bone marrow and splenic supporting cells. The aged G3 Terc ko model faithfully recapitulates the haematopoietic phenotype of aging in humans and we propose to utilise this model to further our understanding of the mechanisms and physiological consequences of clonal hematopoiesis, to eventually help identify strategies to mitigate its clinical consequences.

Platelet CD47 Maintains Circulating Monocyte Immune Homeostasis

In addition to their well-studied hemostatic functions, platelets are immune cells. Platelets circulate at the interface between the vascular wall and white blood cells, and transient platelet-leukocyte complexes are found in both healthy and disease states, positioning platelets to provide physiologic cues of both vascular health and injury. Roles for activated platelets in inducing and amplifying immune responses have received an increasing amount of research attention, but our past studies also showed that normal platelet counts are needed in healthy conditions to maintain immune homeostasis. Circulating monocytes are a part of the first line of host defenses that recognize and clear pathogens in part by releasing inflammatory cytokines, including IL-6, IL-8 and TNFα, that trigger a robust inflammatory reaction. However, this needs to be tightly regulated and in disease conditions like sepsis, dysregulated monocyte responses contribute to a “cytokine storm” that leads to exaggerated inflammation, tissue damage and potentially mortality. The cellular and molecular mechanisms that regulate monocyte immune phenotypes are complex and include roles for platelets. Considering the immune regulatory role of platelets and that thrombocytopenia is associated with worse sepsis outcomes, we hypothesize that resting platelets maintain monocyte immune homeostasis in healthy conditions and thrombocytopenia independently leads to monocyte dysregulation that exacerbates sepsis responses in a manner that is dependent on direct platelet-monocyte CD47 homotypic interactions that regulate monocyte immunometabolism and gene expression. Compared to monocytes from mice with normal platelet counts, circulating monocytes from thrombocytopenic mice had increased toll-like receptor (TLR) induced cytokine responses. Furthermore, ex vivo co-incubation of resting platelets with platelet naïve bone marrow monocytes, induced monocyte metabolic programming skewed toward glycolysis, consistent with our in vitro and in vivo RNA-Seq results. Enhanced glycolysis was mediated by AKT/mTOR pathway activation and resulted in durable changes in TLR agonist responses. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) on monocytes from thrombocytopenic mice showed open chromatin at LPS response genes, and resting platelet interactions with monocytes induced histone H3K9 and H3K27 trimethylation in a CD47 dependent manner. Using mouse models of thrombocytopenia and sepsis, we found that normal platelet numbers limited monocyte immune dysregulation and systemic inflammation. This was dependent on platelet CD47 as only WT platelets, not CD47 -/- platelet transfusion rescued the exaggerated inflammatory responses in thrombocytopenic mice (Fig 1). Both plasma IL-6 and IL-8 and circulating monocyte IL6 and IL8 gene expression from human patients with sepsis and non-septic conditions inversely correlated with patient platelet counts, further suggesting a resting platelet role in maintaining monocyte immune quiescence. Overall, our studies demonstrate that resting platelets maintain monocyte immune homeostasis by limiting TLR responses through regulating monocyte immunometabolic processes that lead to epigenetic changes in monocyte immune response genes (Fig 2). Thrombocytopenia therefore independently leads to immune dysregulation in sepsis, exacerbating systemic inflammatory processes. This is the first demonstration of sterile endogenous cell interactions that regulate innate immune-metabolism and monocyte pathogen responses.

Multiplex Detection of Copy Number Alterations in Multiple Myeloma Including Emerging Therapeutic Targets By Digitalmlpa

Recent advances in immunotherapy have highlighted that in multiple myeloma (MM), besides risk-determining copy number alterations (CNAs) there is a need for investigation of CNAs in CAR-T cell therapy-related targets (e.g. BCMA, GPRC5D, FcRH5). DigitalMLPA technology has been successfully used for characterization of recurrent CNAs on chromosome arms 1p, 1q, 13q and 17p in newly diagnosed and relapsed MM cases, as well as in cancer cell lines of MM origin. Adding probes aimed at CAR-T cell therapy-related target genes complements the already included predictive target probes ( IKZF1/3, IRF4, MYC, RPL5, SLAMF7 and BRAF V600E point mutation) in SALSA® digitalMLPA™ Probemix D006 Multiple Myeloma. In addition, due to a large overlap of affected genomic regions between hematologic neoplasms, we aimed to demonstrate usefulness of this multiplex assay not only on cell lines of MM origin, but also of myeloid and lymphoid lineages. Methods. D006 Multiple Myeloma digitalMLPA probemix contains (i) probes targeting chromosomal arms with recurrent CNAs, including 1p (33 probes), 1q (29 probes), 13q (23 probes), 17p (20 probes); (ii) probes for promising predictive targets such as BCMA, CRBN, GPRC5D, FcRH5, IKZF1/3, IRF4, MYC, RPL5, SLAMF7 - 2-4 probes per gene, and BRAF V600E mutation-specific probe; (iii) 278 probes for subtelomeric, pericentromeric and middle regions of chromosomal arms for all chromosomes; and (iv) control probes for quality control and troubleshooting. DigitalMLPA reactions were performed with 20-40 ng DNA from 29 cancer cell lines of lymphoid and myeloid lineage, 16 of which were of MM origin. Commercial blood-derived genomic DNA samples from healthy individuals were used as references for data normalization using data analysis software Coffalyser digitalMLPA™. Results. Losses of 1p were among the most frequent CNAs in MM cell lines (n=13) and were mainly local (1p21.1-p22.1), including homozygous deletion of CDKN2C in MM cell lines co-occurring with TENT5C subclonal loss in 2 of those. 13q loss (encompassing predominantly the whole arm) was detected in 13 MM cell lines. 1q and 17p losses were present in 12 MM cell lines. One MM cell line showed loss of BCMA (LOPRA-1) and one of CRBN (EJM), 6 had loss of GPRC5D, 9 had MYC gain. None of the MM cell lines carried the BRAF V600E mutation. In the EOL-1 myeloid lineage cell line, trisomy of chromosomes 4, 6, 8 and 19 were correctly identified by increased ratios for all probes covering these chromosomes: 24, 27, 20 and 11 probes respectively; and in the HG-3 lymphoid lineage cell line 11q25 gain and 13q loss was detected. Examples of CNAs detected by D006 Multiple Myeloma probemix are described in Table 1. Copy number ratios of predictive target genes in MM cell lines are shown in Table 2. Ratios between 0.7-1.3 (green cells) indicate normal copy number, ratios below 0.7-1.3 range indicate copy number loss (red cells) and higher ratios indicate gains (blue cells). CNAs detected by digitalMLPA were highly concordant with those reported in public databases. Conclusions. digitalMLPA technology is well suited for multiplex CNA detection of routinely analyzed genomic regions in MM and provides a unique opportunity to research molecular genetic markers of emerging significance in MM in the same reaction. In addition, this MM assay also gives the possibility to analyze gross CNAs in other hematopoietic neoplasms with overlapping affected regions. Input DNA requirements that are a fraction of current WGS, simple protocol and possibility of combining of digitalMLPA libraries with other NGS libraries make D006 Multiple Myeloma digitalMLPA probemix a reliable, cost-effective and robust method to detect well-established and emerging CNAs.

Stathmin-1 Regulates Hematopoietic Stem Cell Mitochondrial and Cellular Function

Mitochondrial function is important to control HSC quiescence and self-renewal. HSCs are known to have low oxidative phosphorylation (OXPHOS), and low mitochondrial reactive oxygen species, however, studies have shown that HSCs have high mitochondrial membrane potential (MMP), which supports their function (Hinge et al 2020; Mansell et al 2021). Microtubules are important for normal mitochondrial function, acting as the main pathway through which mitochondria transport occurs. Stathmin 1 (Stmn1) is a cytoplasmic phosphoprotein that regulates microtubule dynamics via the promotion of microtubule catastrophe or the sequestration of free alpha/beta-tubulin dimers. It is highly expressed in normal hematopoietic stem cells (HSCs) and neural tissues. Also known as oncoprotein 18 (OP18) or leukemia-associated protein 18 (LAP18), Stmn1 is overexpressed in multiple cancers, including both myeloid and lymphoid leukemias. Despite its robust expression in normal HSCs and its elevated expression in leukemias, little is known about Stmn1's role in either normal or malignant HSCs. Herein, we found that Stmn1 is a critical regulator of HSC mitochondrial and cellular function. To determine the role of Stmn1 in HSCs, we first assessed HSC numbers and function in Stmn1 -/-mice. We determined HSC and progenitor numbers, as well as peripheral blood lineages, in young adult Stmn1 -/-mice and WT littermates. Compared to WT, Stmn1 -/- mice have mild anemia and thrombocytopenia, however, HSC numbers are similar. Despite normal HSC numbers, Stmn1 -/-HSCs had markedly impaired repopulating activity in competitive transplantation experiments (10% vs. 60% chimerism at 24 weeks p&amp;lt;0.0001). Similar defects in repopulating activity were found in non-competitive transplants and competitive intra-tibial transplants (done to circumvent any potential homing defects). In addition, Stmn1 -/- HSCs formed fewer colonies when plated in methocult, and were not capable of serial replating. Finally, Stmn1 -/- mice had delayed hematopoietic recovery following a single dose of the chemotherapeutic agent 5-fluorouracil (5-FU). We next assessed the cycling and apoptosis of HSCs. Stmn1 -/- HSCs are hyper-quiescent at baseline, although they can be stimulated to cycle in response to cytokine stimulation (1.3% vs. 3.4% S/G 2/M phase at baseline and 3.4% vs. 7% S/G 2/M phase following stimulation). In addition, they have elevated levels of apoptosis compared to WT (18.5% vs. 9.90%). Consistent with its role as a microtubule destabilizer, Stmn1 expression in HSCs coincides with tubulin expression by confocal immunofluorescent microscopy, and Stmn1 -/- HSCs have decreased tubulin compared to WT HSCs. To gain further insight into their impaired function, we next performed RNA-seq on sorted Stmn1 -/- and WT HSCs. Differential pathway analysis showed alterations in genes associated with cellular metabolism, particularly mitochondrial function (OXPHOS pathway) in the Stmn1 -/- HSCs compared to WT. Indeed, measurement of mitochondrial function using the Seahorse analyzer showed that Stmn1 -/- HSCs and progenitors have impaired maximal respiratory capacity compared to WT (251.29 vs 411.84 pmol/min). In addition, Stmn1 -/- HSCs showed reduced mitochondrial membrane potential and increased mtROS as assessed by flow-based assays. Finally, the mitochondria of Stmn1 -/- HSCs showed markedly abnormal cristae morphology by TEM, consistent with their impaired function, and Stmn1 -/- HSCs have decreased mitochondria number compared to WT (medium 12.8 vs 17.3 per 2D slice cell; p&amp;lt;0.0250). As elevated ROS can impair HSC function, we assessed Stmn1 -/- HSCs following treatment with MitoTEMPO, a specific scavenger of mitochondrial superoxide. Treatment of Stmn1 -/- mice with 4mg/Kg of MitoTEMPO or vehicle control for two weeks resulted in improved HSC colony formation and serial replating capacity in Methocult. Together, these data identify Stmn1 as a critical regulator of HSC function and suggest a model whereby Stmn1 promotes normal microtubule dynamics and mitochondrial health in HSCs. Ongoing experiments are aimed at understanding Stmn1's role in regulating mitochondrial quality control processes such as mitophagy and fission in both normal and malignant HSCs, and in elucidating the effects of Stmn1 loss on leukemic cell growth and viability.

MiR-9-1 Controls Osteoblastic Regulation of Lymphopoiesis

The highly conserved MicroRNA-9 (miR-9) family consists of three members, miR-9-1, miR-9-2 and miR-9-3. These three microRNAs are encoded by unique genes located on different chromosomes, the transcripts of which are processed into the same mature miR-9 sequence. MiR-9 is known to play a crucial role in brain development and neurogenesis, its involvement in hematopoiesis has remained uncertain. To explore this, we investigated the role of miR-9-1 in controlling hematopoiesis. Our findings revealed that miR-9-1 and miR-9-2 but not miR-9-3 were expressed in all hematopoietic cells, with higher levels observed in HSCs, MPPs, and CLPs. In order to understand the impact of miR-9 on hematopoiesis, we employed CRISPR/Cas9-mediated genome editing to create miR-9-1-deficient mice. Deletion of miR-9-1 resulted in reduced mature miR-9 expression, leading to 43% of the mice exhibiting retarded growth and postweaning lethality by 12 weeks of age. The growth-retarded miR-9-1-deficient mice experienced severe lymphopenia, primarily affecting B and T cell development. Further analysis revealed that the populations of pro/pre- and immature B cells in bone marrow (BM) were dramatically reduced in growth-retarded miR-9-1-deficient relative to wild-type control mice. The populations of peripheral T1, T2, FO and MZ B cells were also markedly reduced in growth-retarded miR-9-1-deficient relative to control mice. In addition, the numbers of DN, DP, and SP thymocytes and CD4 + and CD8 + T cells in the spleen were also markedly reduced in growth-retarded miR-9-1-deficient relative to control mice. Consistent with the specific impairment of lymphocyte (B and T cell) development and maturation, the growth-retarded miR-9-1-deficient mice exhibited a reduction of hematopoietic progenitors including ST-HSCs, MPPs, CMPs and especially CLPs, but had normal numbers of GMPs and MEPs. Interestingly, transplantation of miR-9-1-deficient BM cells into wild-type recipients resulted in normal lymphopoiesis, indicating that the lymphopenia was not due to hematopoietic cell autonomous effects but was caused by alterations in the BM stromal microenvironment. We then focused on the effect of miR-9-1 deficiency on the BM niche, particularly the mesenchymal stem cell (MSC) and osteoblastic (OB) niches. The numbers of MSCs in BM stromal cells were comparable between miR-9-1-deficient and wild type mice, whereas the number of osteoblastic cells (OBs) was significantly reduced in the BM stromal cells from growth-retarded miR-9-1-deficient relative to wild-type mice. In agreement with the reduction of OBs in miR-9-1-deficient mice, the mutant MSCs failed to differentiate into OBs. Notably, miR-9-1 deficiency did not influence the differentiation of osteoclasts and adipocytes, suggesting a specific impairment in osteoblastic bone formation. To elucidate the molecular mechanism underlying miR-9-1's regulation of osteoblastic differentiation, we conducted high-throughput RNA sequencing analysis on miR-9-1-deficient MSCs and OBs. The results revealed reduced expression of master transcription factors involved in osteoblastic differentiation, Runt-related transcription factor 2 (Runx2) and Osterix (Osx), and genes related to collagen formation, extracellular matrix organization, and cell adhesion, in miR-9-1-deficient MSCs. Additionally, we identified Follistatin (Fst) as a direct target of miR-9-1. MiR-9-1 deficiency led to increased Fst levels, which, in turn, inhibited BMP signaling in MSCs and reduced IL-7 and IGF-1. In summary, our studies demonstrate that miR-9-1 plays a pivotal role in controlling osteoblastic regulation of lymphopoiesis by targeting the Fst/BMP/Smad signaling axis.

Bone Marrow, Laboratory, and Clinical Features in Pediatric Patients with RUNX1 Familial Platelet Disorder with Associated Myeloid Malignancy (FPDMM)

Background: Germline RUNX1 mutation is associated with an autosomal dominant familial platelet disorder with associated myeloid malignancy (FPDMM), most commonly myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), but lymphoblastic leukemias are also reported. FPDMM is characterized by thrombocytopenia from an early age associated with abnormal platelet function. Herein we report the bone marrow features, in addition to laboratory and clinical findings, of a large pediatric cohort (N=29) with RUNX1-FPDMM. These data have value in guiding early investigation of neonatal/pediatric thrombocytopenia, and baseline bone marrow interpretation to avoid overdiagnosis of MDS or misdiagnosis of immune-mediated thrombocytopenias. Design: The clinical, laboratory, bone marrow pathology, and molecular findings of 29 pediatric patients (&amp;lt;18 years) with germline RUNX1 mutation followed by the natural history study at the National Institutes of Health (NIH) (ClinicalTrials.gov Identifier: NCT03854318), were analyzed. Results : Among the patients, 13 were female and 16 male, in 21 pedigrees, with a median age at bone marrow biopsy of 6 years (range: 17 months to 17 years). Germline RUNX1 mutations were familial in 26 patients and de novo in 3 patients. Easy bruising and propensity for bleeding were common (87%). The median platelet count was 123 x 10 9/L (range: 38 - 244). Abnormal platelet aggregation assay was present in all 9 patients tested. Abnormal transmission electron microscopy of platelets was found in 14/21 (67%) patients tested; the majority had decreased platelet dense granules, with or without decreased alpha granules. Three patients had normal platelet counts. Hematologic neoplasm was identified in 4 patients: one with AML with JAK3 and KRAS mutations; one with B-lymphoblastic leukemia with EBF1::PDGFRB; one with T-lymphoblastic leukemia with a complex karyotype; and one with MDS with excess blasts and no detectable acquired genetic alterations. 3/4 of patients with malignancy had hypercellular marrows. Of the remaining patients, 19/25 (76%) had hypocellular bone marrows for age. The majority had normal numbers of megakaryocytes. Dysmegakaryopoiesis with features overlapping with “dysplasia” (small hypolobated, small with convoluted nuclei and minimal cytoplasm, separation of nuclear lobes) in greater than 10% of megakaryocytes was present in 92% of cases. At least one patient was previously misdiagnosed with immune thrombocytopenic purpura (ITP). 18 patients had eosinophilia in bone marrow sections, six of whom had peripheral blood eosinophilia. Flow cytometric analysis of the marrow commonly showed T-cells with inverted CD4:CD8 ratios (17/22) and increased eosinophils (16/22). The one patient with MDS had increased (10%) immunophenotypically abnormal myeloblasts expressing CD7 and CD123. 23/25 patients had targeted NGS: one patient had a pathogenic mutation in BCOR and the remaining had no pathogenic mutations in genes associated with hematologic malignancy; 15 patients had VUSs reported. At least four patients had large RUNX1 deletions that were not detected by conventional NGS. 23 patients tested had a normal karyotype except one female patient with monosomy X in 11/20 metaphases. Allergy (drug, food and seasonal) was present in 11 patients, and eczema was common. A spectrum of gastrointestinal (GI) symptoms was seen in 11 patients, including constipation, eosinophilic esophagitis, gastroesophageal reflux, and celiac disease. 2 patients were diagnosed with autism, 1 with coarse facies and KDM6B mutation, 1 with brachydactyly syndrome related to PRMT7 mutation, and 1 with non-syndromic hearing loss related to GJB2 mutation. Conclusion: Germline mutation in RUNX1 should be considered in pediatric patients with thrombocytopenia and/or abnormal platelet function and a hypocellular marrow with or without dysmegakaryopoiesis. Dysmegakaryopoiesis in the setting of RUNX1-FPDMM should not be overinterpreted as pediatric MDS without other supporting criteria such as MDS-defining cytogenetic/molecular abnormalities, multilineage dysplasia, or increased blasts. Patients with large deletions in RUNX1 may be missed on routine NGS testing hence proper germline testing in an experienced laboratory is recommended if suspicion is high. Close monitoring is crucial for early identification of emerging myeloid or lymphoid malignancy.

Mice Lacking α-Actinin-1 in Megakaryocytes Display the Feature of Thrombocytopenia and Impair Platelet Functions

Cytoskeleton remodeling and mitochondrial bioenergetics play important roles in platelet biogenesis and functions. α-Actinin-1, a filament crosslinker, was widely expressed in nonmuscle cells, including megakaryocytes (MKs) and platelets. The cytoskeleton is essential for normal mitochondrial morphology, motility, and distribution. In recent years, the accumulated evidence suggests that inherited mutations in α-actinin-1 are implicated in congenital macrothrombocytopenia. However, the role and underlying mechanism of α-actinin-1 in platelet biogenesis and platelet functions remain poorly studied. In this study, we generated an MK-specific α-actinin-1 knockout mouse model (referred to herein as PF4- Actn1 -/-) to investigate the contribution of α-actinin-1 in platelet biogenesis and functions. Blood count tests showed that PF4- Actn1 -/- mice exhibited reduced platelet counts. The decreased platelet number in PF4- Actn1 -/- mice was not attributed to accelerated platelet clearance or impaired TPO generation in vivo. Platelet count changes observed in an anti-GPIbα antibody-induced thrombocytopenia model and splenectomy indicated that the diminished platelet counts in PF4- Actn1 -/- mice might be due to defects in platelet biogenesis. Compared to control mice, PF4- Actn1 -/- mice had normal numbers of MK progenitors, including in LSK, MPP, CMP, GMP, MEP, and PreMegE. H&amp;E staining and flow cytometry revealed a decrease in the number of MKs in BM, but an increase in the number of MKs in the spleen. The absence of α-actinin-1 significantly increased the proportion of 2N-4N MKs and decreased the proportion of 8N-32N MKs. CFU-MK colony formation and MK migration in response to SDF-1 signaling was inhibited in PF4- Actn1 -/- mice. Furthermore, in vitro studies showed a reduced ratio of PPF-bearing MKs in fetal liver-derived PF4- Actn1 -/- MKs. Collectively, these data suggest that α-actinin-1 deficiency in mouse MKs could impair platelet biogenesis, resulting in thrombocytopenia in PF4- Actn1 -/- mice. Platelet adhesion and spreading on immobilized fibrinogen and collagen, thrombin-stimulated clot retraction, and platelet aggregation in response to various concentrations of ADP, thrombin, and collagen was significantly decreased in PF4- Actn1 -/- platelets. PF4- Actn1 -/- platelets also exhibited decreased integrin αIIbβ3 activation and reduced P-selectin exposure in response to various concentrations of ADP, ADP+EPI, thrombin, collagen, and convulxin. Notably, PF4- Actn1 -/- platelets showed inhibited calcium mobilization, reactive oxygen species (ROS) generation, and actin polymerization in response to collagen and thrombin. Furthermore, PF4- Actn1 -/- mice demonstrated significantly impaired hemostasis. No differences were observed in the PT, APTT, TT, and fibrinogen concentrations between PF4- Actn1 -/- mice and Actn1 f/f mice. However, tail, liver, and brain bleeding tests revealed that PF4- Actn1 -/- mice had significantly prolonged bleeding time and increased bleeding volume. Kaolin-activated whole thromboelastography (TEG) mapping assay indicated that there were no differences in the R-time, K-time, and α-angle values, but the maximum amplitude was decreased in PF4- Actn1 -/- mice. To explore the mechanism of α-actinin-1 in platelet biogenesis and platelet functions. Low- (2-4N) and high-ploidy (≥8N) Actn1 f/f and PF4- Actn1 -/- MKs were sorted for quantitative proteomics analysis. The results of 4D label-free quantitative proteomics revealed that protein expression was significantly decreased in high-ploidy MKs following α-actinin-1 knockdown. Functional enrichment analysis of differentially expressed proteins indicated that α-actinin-1 deletion reduced platelet activation and mitochondrial functions. PF4- Actn1 -/- platelets exhibited reduced mitochondrial membrane potential, mitochondrial ROS generation, mitochondrial respiration and glycolysis, suggesting alterations in mitochondrial function in platelets. In this study, we report that mice with α-actinin-1 deficiency in MKs recapitulate the features of thrombocytopenia and exhibit impaired platelet functions.

Conditional Oprk1-dependent Kiss1 deletion in kisspeptin neurons caused estrogen-dependent LH pulse disruption and LH surge attenuation in female rats

The gonadotropin-releasing hormone (GnRH) pulse and surge are considered to be generated by arcuate kisspeptin/neurokinin B/dynorphin A (KNDy) neurons and anteroventral periventricular nucleus (AVPV) kisspeptin neurons, respectively, in female rodents. The majority of KNDy and AVPV kisspeptin neurons express κ-opioid receptors (KORs, encoded by Oprk1) in female rodents. Thus, this study aimed to investigate the effect of a conditional Oprk1-dependent Kiss1 deletion in kisspeptin neurons on the luteinizing hormone (LH) pulse/surge and fertility using Kiss1-floxed/Oprk1-Cre rats, in which Kiss1 was deleted in cells expressing or once expressed the Oprk1/Cre. The Kiss1-floxed/Oprk1-Cre female rats, with Kiss1 deleted in a majority of KNDy neurons, showed normal puberty while having a one-day longer estrous cycle and fewer pups than Kiss1-floxed controls. Notably, ovariectomized (OVX) Kiss1-floxed/Oprk1-Cre rats showed profound disruption of LH pulses in the presence of a diestrous level of estrogen but showed apparent LH pulses without estrogen treatment. Furthermore, Kiss1-floxed/Oprk1-Cre rats, with Kiss1 deleted in approximately half of AVPV kisspeptin neurons, showed a lower peak of the estrogen-induced LH surge than controls. These results suggest that arcuate and AVPV kisspeptin neurons expressing or having expressed Oprk1 have a role in maintaining normal GnRH pulse and surge generation, the normal length of the estrous cycle, and the normal offspring number in female rats.

Novel Method for Ranking Generalized Fuzzy Numbers Based on Normalized Height Coefficient and Benefit and Cost Areas

This paper proposes a method for ranking generalized fuzzy numbers, which guarantees that both horizontal and vertical values are important parameters affecting the final ranking score. In this method, the normalized height coefficient is introduced to evaluate the influence of the height of fuzzy numbers on the final ranking score. The higher the normalized height coefficient of a generalized fuzzy number is, the higher its ranking. The left and right areas are presented to calculate the impact of the vertical value on the final ranking score. The left area is considered the benefit area. The right area is considered the cost area. A generalized fuzzy number is preferred if the benefit area is larger and the cost area is smaller. The proposed method can be employed to rank both normal and non-normal fuzzy numbers without normalization or height minimization. Numerical examples and comparisons with other methods highlight the feasibility and robustness of the proposed method, which can overcome the shortcomings of some existing methods and can support decision-makers in selecting the best alternative.

Screening for a practical method to monitor the status of patients with metastatic bladder cancer at the circulating cell-gene level

Identifying a novel method to monitor metastatic bladder cancer status at the cell-gene level could lead to earlier appropriate therapeutic intervention and better outcomes. In this study, we evaluated a practical method to monitor the cancer status at the circulating cell-gene level before and after treatment in fourteen patients with metastatic bladder cancer who were indicated for systemic drug therapy. Patients were assessed via imaging before and after drug treatment, and cell-free DNA (cfDNA) analysis was performed to detect three parameters: cfDNA level, ERRB2 gene copy numbers, and telomerase reverse transcriptase (TERT) gene mutations. We hypothesized that decreased cfDNA levels, a normal copy number of ERB-B2 receptor tyrosine kinase 2 (ERBB2), and the absence of the TERT C228T mutation indicate cancer suppression. We found that a > 1.8-fold increase in cfDNA levels, increased copy number of ERBB2, or the existence of the TERT C228T mutation indicated disease progression. Stable cfDNA levels, normal ERBB2 copy number, and the absence of TERT C228T mutations indicate a stable cancer status. Collectively, our results show that the combination of cfDNA concentration, TERT mutation, and ERBB2 copy number may be useful for determining the efficacy of drug therapy in patients with metastatic bladder cancer.