Proliferation Centers Research Articles

Maf expression in B cells restricts reactive plasmablast and germinal center B cell expansion

Precise regulation of B cell differentiation is essential for an effective adaptive immune response. Here, we show that B cell development in mice with B cell-specific Maf deletion is unaffected, but marginal zone B cells, germinal centre B cells, and plasmablasts are significantly more frequent in the spleen of naive Maf-deficient mice compared to wild type controls. In the context of a T cell-dependent immunization, Maf deletion causes increased proliferation of germinal centre B cells and extrafollicular plasmablasts. This is accompanied by higher production of antigen-specific IgG1 antibodies with minimal modification of early memory B cells, but a reduction in plasma cell numbers. Single-cell RNA sequencing shows upregulation of genes associated with DNA replication and cell cycle progression, confirming the role of Maf in cell proliferation. Subsequent pathway analysis reveals that Maf influences cellular metabolism, transporter activity, and mitochondrial proteins, which have been implicated in controlling the germinal centre reaction. In summary, our findings demonstrate that Maf acts intrinsically in B cells as a negative regulator of late B cell differentiation, plasmablast proliferation and germinal centre B cell formation.

REACTION OF LYMPHOID TISSUE IN THE WALL OF THE DUODENUM AND LYMPHOID PLAQUE IN RATS IN MODELING HYPOKINESIA

The damaging effect of hypokinesia on the functional state of lymphoid tissue in the wall of the duodenum and on the lymphoid plaque was established. It was found that the processes of cell destruction, suppression of the plasma reaction and blast transformation of cells are most pronounced in the wall of the duodenum. In the lymphoid plaque, along with a high level of destructive processes in lymphoid cells, lymphocytopoiesis is preserved in the centers of proliferation of lymphoid nodules and the proportion of plasma cells increases in the internodal zone, which indicates the development of compensatory processes under the long-term influence of hypokinesia.

Assessment for acceleration and transformation of chronic lymphocytic leukemia/small lymphocytic lymphoma using histologic and immunohistochemical features: a case series

Morphologic features of aggressive/ “accelerated” chronic lymphocytic leukemia/small lymphocytic lymphoma (aCLL/SLL) have been described. Richter transformation (RT) also occurs in a subset of CLL/SLL cases. This case series examined inter-observer variability when assessing for aCLL/SLL and RT, with attention to how immunohistochemical (IHC) markers may assist in this evaluation. Twelve cases of CLL/SLL with available FFPE tissue were identified. H&E staining and IHC (CD3, CD20, CD5, CD23, LEF1, LAG3, C-MYC, PD-1, MUM1, Cyclin D1, BCL-6, p53, and Ki-67) were performed. Three hematopathologists reviewed each case. The pathologists provided a final interpretation of (1) CLL/SLL, (2) CLL/SLL with expanded and/or confluent proliferation centers or increased Ki-67 (aCLL/SLL), or (3) large cell transformation/DLBCL. The pathologists lacked consensus in the diagnosis in 6/12 cases (50%). The reviewers disagreed on the presence of expanded/confluent proliferation centers in 8/12 cases (67%). With the exception of Ki-67, no IHC marker showed a difference in the staining profile in aCLL/SLL or RT compared to low-grade cases. This series showed inter-observer variability in the evaluation for aCLL/SLL and RT. A study that serially examines genetic alterations in FFPE tissue and correlates the features with histology and IHC, at diagnosis and throughout the disease course, may help refine indicators of aggressive disease.

Immediate but Temporal Response: The Role of Distal Epithelial Cells in Wound Healing.

Efficient oral mucosal wound healing requires coordinated responses from epithelial progenitor cells, yet their spatiotemporal recruitment and activation remain unclear. Using a mouse model of palatal mucosal wound healing, we investigated the dynamics of epithelial cells during this process. Proliferation analysis revealed that, in addition to the expected proliferation center near the wound edge, distal cell populations rapidly activated post-injury by elevating their mitotic activity. These distal cells displayed predominant lateral expansion in the basal layer, suggesting roles beyond just tissue renewal. However, while proximal proliferation center cells sustained heightened proliferation until re-epithelialization was completed, distal cells restored basal turnover rates before wound closure, indicating temporally confined contributions. Lineage tracing of Wnt-responsive epithelial cells showed remarkable clone expansion in basal layers both proximally and distally after wounding, contrasting with gradual clone expansion in homeostasis. Although prioritizing tissue repair, epithelial progenitor cells maintained differentiation programs and barrier functions, with the exception of the leading edge. At the leading edge, we found accelerated cell turnover, but the differentiation program was suspended. In summary, our findings uncovered that oral wound re-epithelialization involves two phases: an initial widespread response with proliferation of proximal and distal cells, followed by proliferation confined to the wound proximal region. Uncovering these stage-specific healing mechanisms provides insights for developing targeted therapeutic strategies to improve wound care.

Spatial patterning controls neuron numbers in the Drosophila visual system

Neurons must be made in the correct proportions to communicate with the appropriate synaptic partners and form functional circuits. In the Drosophila visual system, multiple subtypes of distal medulla (Dm) inhibitory interneurons are made in distinct, reproducible numbers-from 5 to 800 per optic lobe. These neurons are born from a crescent-shaped neuroepithelium called the outer proliferation center (OPC), which can be subdivided into specific domains based on transcription factor and growth factor expression. We fate mapped Dm neurons and found that more abundant neural types are born from larger neuroepithelial subdomains, while less abundant subtypes are born from smaller ones. Additionally, morphogenetic Dpp/BMP signaling provides a second layer of patterning that subdivides the neuroepithelium into smaller domains to provide more granular control of cell proportions. Apoptosis appears to play a minor role in regulating Dm neuron abundance. This work describes an underappreciated mechanism for the regulation of neuronal stoichiometry.

Clinicopathological Study of PD-1/PD-L1 Expression in Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (CLL/SLL) with Emphasis on Large B-Cell Richter Transformation.

In the era of immunotherapy, inhibition of the PD-1/PD-L1 immune checkpoint pathway has changed the therapeutic landscape for many tumors. Limited studies were performed on the expression of PD-1 in chronic lymphocytic lymphoma/small lymphocytic lymphoma (CLL/SLL) and its Richter transformation into diffuse large B-cell lymphoma (DLBCL-RT). This study aims to evaluate PD-1/PD-L1 expression and their prognostic role in CLL/SLL, DLBCL-RT, and DLBCL-de novo patients. This retrospective study was conducted on 96 cases (38 CLL/SLL, 11 DLBCL-RT and 47 DLBCL-de novo) that were retrieved from the pathologic and clinical databases at the Oncology Center, Mansoura University. Immunohistochemical evaluation of PD-1 and PD-L1 was assessed in tumor cells and the microenvironment in those patients. This study demonstrated positive expression of PD-1 in CLL/SLL patients, mainly in proliferation centers. Moreover, it showed a higher prevalence of PD-1 expression in DLBCL-RT (9/11 patients) than in DLBCL-de novo (5/47 patients) (P < 0.001). Tumor cells revealed positive PD-L1 expression in 5/47 DLBCL-de novo patients and negative PD-L1 expression in all CLL/SLL and DLBCL-RT patients. PD-1 was positive in reactive T-cells, and PD-L1 was positive in background histiocytes and dendritic cells in all studied cases. PD-1 positive expression in tumor cells was considered an independent poor prognostic factor for overall survival (OS) in DLBCL patients (P = 0.04). In addition, DLBCL-RT had a significantly shorter OS than DLBCL-de novo (P = 0.005). The high prevalence of PD-1 expression in DLBCL-RT patients supports the promising and potential role of anti-PD-1 immunotherapy in the treatment of DLBCL-RT patients.

Investigation into Intraclonal Heterogeneity of CXCR4 DimCD5 Bright Chronic Lymphocytic Leukemia Cells Identifies Distinct Activation Signatures

Chronic Lymphocytic Leukemia (CLL) is a heterogeneous disease associated with diverse clinical and molecular profiles, suggesting long-term clonal evolution driven by genetic diversification from a small, often undetected, subset of leukemic cells endowed with superior growth capacities. More importantly, this diversification process enhances the opportunity of CLL subclones to survive selective pressure, such as BTK or PLCG2 mutations that can abolish the effect of BTK inhibitors. While this evolutionary process is inevitable in leukemic cells, therapeutic intervention targeting the cells that have just replicated their DNA, and hence could have accumulated new genomic mutation, would be an effective strategy to significantly delay or abort the emergence of deleterious mutations. In CLL, a series of studies involving consumption of 2H 2O determined that CXCR4 DimCD5 Bright cells (proliferative fraction, PF) of CLL clones contain most of the recently divided cells, identified by the presence of 2H-DNA. This conclusion was supported by finding elevated numbers of Ki67 + and MCM6 + cells in the same fraction, when compared to other CLL cells. Furthermore, PF is superior in: lymphoid tissue homing in xenograft models; activating T cells; the capacity to alter tumor microenvironment (TME) by Th2 skewing; and expression of AICDA which can increase the likelihood of off-target genomic mutations. To permit a focused analysis of PF that would allow us to intercept this evolutionary loop, we characterized the single cell transcriptomes of PF from 4 U-CLL and 4 M-CLL patients immediately prior to ibrutinib treatment. Consistent with previous reports, the majority of PF (herein APC-PF) cells express certain antigen presentation molecules with a gene signature suggestive of T cell regulation and cell-cell interactions. Additionally, we discovered a small subpopulation with an upregulation of MIR155HG, HSP90AB, CCL3, CCL4, MYC and a concomitant MYC activation signature (herein MYC-PF). Since miR-155 expression can be induced by CD40L or B-cell-activating factors, this PF subset may reflect prior BCR ligation and/or T cell co-stimulation. However, the strongest BCR activation signature was found in another PF subpopulation (herein BCR-PF) with an enrichment of germinal center-related genes (e.g., BACH2, PAX5, FOXP1) and CLL-associated molecules ( BCL2, ROR1, MSI2, PLCG2). Furthermore, the BCR-PF cells are least likely to be resting (in G1/G0) compared to others. Collectively, these findings suggest effective BCR engagement, either from the TME or cell-autonomously. Corroborating these findings with our previous studies documenting the CXCR4/CD5 fractions and the expression of protein proliferation markers, our transcriptomic analyses suggest that the BCR-PF may be the most immediate emigrant from the proliferation centers (PC) within CLL lymph nodes (LN). To probe this possibility, spatial transcriptomic analysis was performed on FFPE LN tissues from surgical biopsies from 3 U-CLL and 1 M-CLL patients. Single cell deconvolution was used to determine the relative abundance of cells within the anatomically defined PCs that resemble the three PF subpopulations transcriptomically. Our results suggest that PCs are more enriched in cells resembling BCR-PF, followed by cells bearing the APC-PF signature. To extend on these observations, we have documented that the unique surface markers expressed in these PF subfractions positively correlate with our transcriptomic results. Taken together, these results not only highlighted the molecular heterogeneity of CLL, but also lead to the identification of two PF subpopulations that may provide novel actionable therapeutic targets to significantly halt CLL clonal evolution.

CLL Cells within the Proliferation Centers of the Patient Lymph Nodes Are Enriched for Notch Signaling, Thus NOTCH a Viable Target for CLL Therapy

CLL cells within the Proliferation Centers of the Patient Lymph Nodes are enriched for Notch signaling, thus a Target for CLL Therapy. Shantaram S. Joshi, Michael Pitner, Matt Kling, Furqan Mehdi, Mamta Shukla, Ashima Shukla, Vipul Shukla, Nagendra Chaturvedi, Hamid Band , Javeed Iqbal The tissue microenvironment, particularly the lymph node (LN), plays a major role in regulating the leukemic progression and clinical heterogeneity of chronic lymphocytic leukemia (CLL). CLL cells proliferate in the patient lymph nodes, resulting in the formation of proliferation centers (PCs). The molecular basis of the CLL cell proliferation within PCs is not well known. Several reports have implicated the activated Notch signaling pathway in the pathogenesis of CLL. Using the IRF4 -/-Vh11 mouse model of CLL, we have previously shown that Notch signaling is indispensable for CLL development (Vipul Shukla et al, Oncotarget, 2016). Based on these studies, we hypothesized that CLL cell proliferation in the lymph node PCs involves activated NOTCH signaling. To this end, we have examined the expression levels of Notch signaling associated molecules namely IRF4, Notch 1, Notch 2, Nedd4, Fbxw7 and Itch1 on CLL cells within the patient lymph node proliferation centers (n=12) using immunohistochemistry and western blotting. Expression levels were scored based on the percentage of cells with positive staining: 5+ (&amp;gt;80%), 4+ (60-80%), 3+ (40-60%), 2+ (20-40%), 1+ (&amp;lt;20%) and 0 (No staining). IHC analyses showed that the expression levels of IRF4 to be 2+, Notch 1 was 4+, Notch 2 was -3 to 4+, Nedd4 was 1+, Fbxw7 was 3+ and ITCH1 was 1+. Western blotting of CLL cells from the lymph node proliferation centers (n=6) confirmed a similar pattern of expression of the Notch-associated genes. Together, these results validate the association of Notch signaling in CLL cells of the patient lymph node PCs similar to CLL cells from the IRF4 -/-Vh11 mouse model. Therefore, as a next logical step we treated the MEC-1 and OSU-CLL cells in culture and primary CLL cell from patients with NOTCH inhibitor RO4929097 (RO), a γ-secretase inhibitor for 24, 48 and 72 hours in vitro. There was a significant decrease in the expression NOTCH 1, NOTCH 2 in MEC-1, OSU-CLL and primary CLL cells determined using western blot technique. Also, we observed a significant decrease in proliferation of CLL cells from MEC-1 and OSU-CLL cell lines, following treatment with NOTCH inhibitor RO. In addition, NOTCH inhibitor combined with Bruton's Tyrosine Kinase (BTK) a member of the B-Cell Receptor signaling, showed a further significant decrease in the proliferation of CLL cells in culture. Together these experiments demonstrated that NOTCH targeting strategy is a viable therapy for aggressive CLL warranting further preclinical and clinical studies. This work was partially supported by bridge funding from the American Association of Hematology awarded to Dr. Runqing Lu, who passed away during the pursuit of this study.

The Role of the Microenvironment and Cell Adhesion Molecules in Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is a B-cell malignancy whose progression largely depends on the lymph node and bone marrow microenvironment. Indeed, CLL cells actively proliferate in specific regions of these anatomical compartments, known as proliferation centers, while being quiescent in the blood stream. Hence, CLL cell adhesion and migration into these protective niches are critical for CLL pathophysiology. CLL cells are lodged in their microenvironment through a series of molecular interactions that are mediated by cellular adhesion molecules and their counter receptors. The importance of these adhesion molecules in the clinic is demonstrated by the correlation between the expression levels of some of them, in particular CD49d, and the prognostic likelihood. Furthermore, novel therapeutic agents, such as ibrutinib, impair the functions of these adhesion molecules, leading to an egress of CLL cells from the lymph nodes and bone marrow into the circulation together with an inhibition of homing into these survival niches, thereby preventing disease progression. Several adhesion molecules have been shown to participate in CLL adhesion and migration. Their importance also stems from the observation that they are involved in promoting, directly or indirectly, survival signals that sustain CLL proliferation and limit the efficacy of standard and novel chemotherapeutic drugs, a process known as cell adhesion-mediated drug resistance. In this respect, many studies have elucidated the molecular mechanisms underlying cell adhesion-mediated drug resistance, which have highlighted different signaling pathways that may represent potential therapeutic targets. Here, we review the role of the microenvironment and the adhesion molecules that have been shown to be important in CLL and their impact on transendothelial migration and cell-mediated drug resistance. We also discuss how novel therapeutic compounds modulate the function of this important class of molecules.

A novel proneural function of Asense is integrated with the sequential actions of Delta-Notch, L'sc and Su(H) to promote the neuroepithelial to neuroblast transition.

In order for neural progenitors (NPs) to generate distinct populations of neurons at the right time and place during CNS development, they must switch from undergoing purely proliferative, self-renewing divisions to neurogenic, asymmetric divisions in a tightly regulated manner. In the developing Drosophila optic lobe, neuroepithelial (NE) cells of the outer proliferation center (OPC) are progressively transformed into neurogenic NPs called neuroblasts (NBs) in a medial to lateral proneural wave. The cells undergoing this transition express Lethal of Scute (L'sc), a proneural transcription factor (TF) of the Acheate Scute Complex (AS-C). Here we show that there is also a peak of expression of Asense (Ase), another AS-C TF, in the cells neighboring those with transient L'sc expression. These peak of Ase cells help to identify a new transitional stage as they have lost NE markers and L'sc, they receive a strong Notch signal and barely exhibit NB markers. This expression of Ase is necessary and sufficient to promote the NE to NB transition in a more robust and rapid manner than that of l'sc gain of function or Notch loss of function. Thus, to our knowledge, these data provide the first direct evidence of a proneural role for Ase in CNS neurogenesis. Strikingly, we found that strong Delta-Notch signaling at the lateral border of the NE triggers l'sc expression, which in turn induces ase expression in the adjacent cells through the activation of Delta-Notch signaling. These results reveal two novel non-conventional actions of Notch signaling in driving the expression of proneural factors, in contrast to the repression that Notch signaling exerts on them during classical lateral inhibition. Finally, Suppressor of Hairless (Su(H)), which seems to be upregulated late in the transitioning cells and in NBs, represses l'sc and ase, ensuring their expression is transient. Thus, our data identify a key proneural role of Ase that is integrated with the sequential activities of Delta-Notch signaling, L'sc, and Su(H), driving the progressive transformation of NE cells into NBs.

Sialylation regulates migration in chronic lymphocytic leukemia.

Sialylation is the terminal addition of sialic acid to underlying glycans. It plays a prominent role in cell adhesion and immune regulation. Sialylated structures found on adhesion molecules, such as CD49d, mediate the interactions between cancer cells and the microenvironment, facilitating metastatic seeding in target organs. Chronic lymphocytic leukemia (CLL) is a clonal B-cell malignancy characterized by the accumulation of CD5-positive B cells in the peripheral blood, bone marrow and lymph nodes. CLL cells proliferate mainly in the lymph node "proliferation centers", where the microenvironment provides pro-survival signals. Thus, migration and homing into these protective niches play a crucial role in CLL biology. In recent years, therapeutic strategies aimed at inducing the egress of CLL cells from the lymph nodes and bone marrow into the circulation have been highly successful. In this study, the sialylation status of 79 untreated and 24 ibrutinib-treated CLL patients was characterized by flow cytometry. Moreover, the effect of sialic acid removal on migration was tested by a transwell assay. Finally, we examined the sialylation status of CD49d by Western blot analysis. We found that CLL cells are highly sialylated, particularly those characterized by an "activated" immune phenotype. Notably, sialylation regulates CLL migration through the post-translational modification of CD49d. Finally, we showed that therapeutic agents that induce CLL mobilization from their protective niches, such as ibrutinib, modulate sialic acid levels. We propose that sialylation is an important regulator of CLL trafficking and may represent a novel target to further improve CLL therapy.

CRISPR-Cas9 Genome Editing of Primary Chronic Lymphocytic Leukemia Cells

Chronic lymphocytic leukemia (CLL) is a common B-cell neoplasia that exhibits a very heterogeneous course. Over the last decade, multiple molecular studies have described the genomic, epigenomic and transcriptional landscape of the disease. These studies have led to the identification of more than 60 recurrently mutated genes as well as a large number of deregulated genes and regulatory elements. While the prognostic significance of some of these alterations is well described, their specific contributions to the pathogenesis of the disease remains largely unknown. Attempts to address this experimentally have employed genetically engineered mouse models and human cell lines. Although mouse models have been helpful to recapitulate disease aspects that can be only properly studied in vivo, significant limitations exist that prevent extrapolation of data from mice to human. Moreover, cell lines are commonly obtained from patients with end-stage, refractory diseases and are frequently EBV-positive, showing great discrepancies with primary cells, including high proliferation rate and absence of spontaneous apoptosis. In addition, they do not faithfully recapitulate the transcriptional and chromatin landscape of CLL primary cases. Thus, there is a need of better cellular models that recapitulate these disease aspects. An attractive solution to this problem would be the use of primary human CLL cells as a platform for ex vivo genetic manipulation, applying CRISPR-Cas9 genome editing technology. However, this is technically challenging, as (i) CLL cells are critically dependent on stimulatory signals coming from the tumor microenvironment, particularly in the lymph nodes (LN), and (ii) primary cells are resistant to most gene transfer methods. In order to overcome these limitations, we took advantage of a cell culture system that is reminiscent of a LN environment and induces vigorous proliferation of CLL primary cells for several weeks, allowing retroviral gene transfer with high efficiency and minimal toxicity (Mangolini, M. et al., 2022, Nat Commun, in press). To avoid undesired off-target effects induced by the constitutive expression of a CRISPR-Cas9 viral plasmid, we used transient expression systems. As an initial proof of concept, we designed sgRNA targeting the CD19 gene, a pan-B cell marker highly expressed in CLL cells, and assessed the transfection efficiency and cell viability when introducing the Cas9-CD19 sgRNA ribonucleoprotein complex using the Neon Transfection System (Thermo Fisher Scientific). Cryopreserved CLL primary cells (from peripheral blood samples) were expanded for 3 days in vitro and were subsequently electroporated with optimized conditions. Cell viability and electroporation efficiency were assessed 24 hours later and demonstrated a remarkable low percentage of apoptotic cells, with only 10% of cell death compared to non-electroporated cells (measured by flow cytometry using the LIVE/DEAD fixable Aqua dead cell stain, n= 6 cases); while high transfection efficiency was accomplished, with over 80% of cells containing the fluorescent Cas9-ATTO550 sgRNA ribonucleoprotein. Finally, 72 hours after electroporation we observed a downregulation greater than 85% of CD19 levels by flow cytometry (n=6 cases). After successful CD19 downregulation, we aimed at targeting the cell cycle regulatory protein Cyclin D2 (CCND2), which is upregulated by CLL cells at proliferation centers and the most upregulated cyclin in our cell culture system. Two independent sgRNAs were electroporated with the Cas9 protein into 3 CLL primary cases, with a minimum effect on cell viability after 24 hours. CCND2 knock-down (KD) efficiency was of 70% and 60% respectively in all cases studied, assessed by qRT-PCR. Interestingly, despite being exposed to cell culture conditions promoting vigorous proliferation, CCND2 KD blocked cell proliferation, and a cell growth reduction of 50-70% was obtained with both sgRNAs compared with non-targeting controls already at 48 hours. Altogether, these results demonstrate the feasibility to genetically modify primary CLL cells using the CRISPR-Cas9 system. This strategy overcomes the current dependency on cell lines or mouse models and represents a powerful tool to unveil the role of candidate genes involved in CLL pathogenesis.

Idiopathic Multicentric Castleman Disease Interactive Disease Awareness and Educational Tool for Pathologists

Background: Idiopathic multicentric Castleman disease (iMCD) is a rare systemic lymphoproliferative disease that is challenging to diagnose. Diagnostic criteria require the identification of characteristic histopathology in the context of supportive clinical, radiologic, and serologic findings. However, the histologic features of iMCD can be subtle and nonspecific, which delays an accurate diagnosis. Timely diagnosis and early intervention are essential to reduce patient mortality. Developing a disease awareness and educational tool for pathologists may support the identification of future iMCD cases. Methods: International pathologists were identified to support the development of an interactive pathology tool for iMCD. To validate the tool, a selection of pathologists attended a virtual workshop to provide insight and help shape the content and format of the tool. Prior to workshop, attendees were asked to complete a 14-question survey on the diagnosis of iMCD, including exclusion of other diseases, conditions with similar histopathological features, and features specific to iMCD. Results: The educational tool is currently in development and takes into consideration the varied diagnostic journeys in different centers/countries. Histopathologic criteria for iMCD require evaluation of the degree of plasmacytosis, regressed germinal centers, follicular dendritic cell proliferation, vascular proliferation, and hyperplastic germinal centers. While the differences between high grade (grade 3) and normal (grade 0) findings are straightforward, differentiating grade 1 from grade 2 (the diagnostic cut-off) is challenging and subjective. The tool will focus on these diagnostic grey-zones to include points of differentiation between Grades 1 and 2 iMCD, disease exclusion criteria, and the use of diagnostic images that can be explored. Thus far, development of the tool has shown there is a need for standardized disease terminology between pathologists, that the use of template reports and language guidance may support the diagnostic process, and that patient case studies and the use of real-world examples support disease recognition. The educational tool is anticipated to be ready for release in September 2022. Conclusions: Diagnosis of iMCD is complex, and, despite the existence of well-established diagnostic criteria, pathological differentiation remains a challenge. Increasing awareness of iMCD-specifically the key diagnostic criteria-will assist with both timely diagnoses and the treatment of this disease.

Spalt and disco define the dorsal-ventral neuroepithelial compartments of the developing Drosophila medulla.

Spatial patterning of neural stem cell populations is a powerful mechanism by which to generate neuronal diversity. In the developing Drosophila medulla, the symmetrically dividing neuroepithelial cells of the outer proliferation center crescent are spatially patterned by the nonoverlapping expression of 3 transcription factors: Vsx1 in the center, Optix in the adjacent arms, and Rx in the tips. These spatial genes compartmentalize the outer proliferation center and, together with the temporal patterning of neuroblasts, act to diversify medulla neuronal fates. The observation that the dorsal and ventral halves of the outer proliferation center also grow as distinct compartments, together with the fact that a subset of neuronal types is generated from only one half of the crescent, suggests that additional transcription factors spatially pattern the outer proliferation center along the dorsal-ventral axis. Here, we identify the spalt (salm and salr) and disco (disco and disco-r) genes as the dorsal-ventral patterning transcription factors of the outer proliferation center. Spalt and Disco are differentially expressed in the dorsal and ventral outer proliferation center from the embryo through to the third instar larva, where they cross-repress each other to form a sharp dorsal-ventral boundary. We show that hedgehog is necessary for Disco expression in the embryonic optic placode and that disco is subsequently required for the development of the ventral outer proliferation center and its neuronal progeny. We further demonstrate that this dorsal-ventral patterning axis acts independently of Vsx1-Optix-Rx and thus propose that Spalt and Disco represent a third outer proliferation center patterning axis that may act to further diversify medulla fates.

Artificial intelligence-assisted mapping of proliferation centers allows the distinction of accelerated phase from large cell transformation in chronic lymphocytic leukemia

AbstractChronic lymphocytic leukemia/small lymphocytic lymphoma (CLL) is characterized morphologically by numerous small lymphocytes and pale nodules composed of prolymphocytes and paraimmunoblasts known as proliferation centers (PCs). Patients with CLL can undergo transformation to a more aggressive lymphoma, most often diffuse large B-cell lymphoma (DLBCL), known as Richter transformation (RT). An accelerated phase of CLL (aCLL) also may be observed which correlates with subsequent transformation to DLBCL, and may represent an early stage of transformation. Distinguishing PCs in CLL from aCLL or RT can be diagnostically challenging, particularly in small needle biopsy specimens. Available guidelines pertaining to distinguishing CLL from its' progressive forms are limited, subject to the morphologist's experience and are often not completely helpful in the assessment of scant biopsy specimens. To objectively assess the extent of PCs in aCLL and RT, and enhance diagnostic accuracy, we sought to design an artificial intelligence (AI)-based tool to identify and delineate PCs based on feature analysis of the combined individual nuclear size and intensity, designated here as the heat value. Using the mean heat value from the generated heat value image of all cases, we were able to reliably separate CLL, aCLL and RT with sensitive diagnostic predictive values.

Use of Bidirectional Long Short Term Memory in Spoken Word Detection with reference to the Assamese language

Objectives : The proposed method is based on a unique technique of Deep learning for identifying spoken words with reference to Assamese language. Most of the DNN based algorithms have been successfully implemented in the field of image recognition, computer vision, natural language processing and medical picture analysis. Methods: The method used here is the Bidirectional Long Short Term Memory (BLSTM). BLSTM incorporates both past and future situations together. The speech database for this research work is hired from the repository of Indian Language Technology Proliferation and Development Center (ILTP-DC). This repository contains 32,335 utterances by 1000 numbers of male and female participants, which is comprised of 262 unique Assamese native words. The BLSTM based recognition model is using 10 out of the 262 unique words and the remaining words are used in construction or generation of synthesized sentences. The feature extraction module uses 39 feature coefficients, which are composed of MFCC, ΔMFCC and ΔΔMFCC coefficients. Findings: The Word Error Rate (WER) of the BLSTM based recognition model is 18.84% with an average accuracy of 98.12%, which sets one promising benchmark when compared to recent findings. Novelty: In this work an attempt has been made with a different approach to detect certain keywords of Assamese language by adopting deep learning methodology. The future objective of this proposed work is to improve the detection capability of this model by considering multiple DNN models together in a hybrid approach along with the inclusion of additional features. Keywords: Bidirectional Long Short Term Memory; Deep Learning; Speech recognition; WER; MFCC

B-cell Receptor Signaling Induced Metabolic Alterations in Chronic Lymphocytic Leukemia Can Be Partially Bypassed by TP53 Abnormalities.

It has been unclear what role metabolism is playing in the pathophysiology of chronic lymphocytic leukemia (CLL). One reason is that the study of CLL metabolism is challenging due to the resting nature of circulating CLL cells. Also, it is not clear if any of the genomic aberrations observed in this disease have any impact on metabolism. Here, we demonstrate that CLL cells in proliferation centers exhibit upregulation of several molecules involved in glycolysis and mitochondrial metabolism. Comparison of CXCR4/CD5 intraclonal cell subpopulations showed that these changes are paralleled by increases in the metabolic activity of the CXCR4lowCD5high fraction that have recently egressed from the lymph nodes. Notably, anti-IgM stimulation of CLL cells recapitulates many of these metabolic alterations, including increased glucose uptake, increased lactate production, induction of glycolytic enzymes, and increased respiratory reserve. Treatment of CLL cells with inhibitors of B-cell receptor (BCR) signaling blocked these anti-IgM-induced changes in vitro, which was mirrored by decreases in hexokinase 2 expression in CLL cells from ibrutinib-treated patients in vivo. Interestingly, several samples from patients with 17p-deletion manifested increased spontaneous aerobic glycolysis in the unstimulated state suggestive of a BCR-independent metabolic phenotype. We conclude that the proliferative fraction of CLL cells found in lymphoid tissues or the peripheral blood of CLL patients exhibit increased metabolic activity when compared with the bulk CLL-cell population. Although this is due to microenvironmental stimulatory signals such as BCR-engagement in most cases, increases in resting metabolic activity can be observed in cases with 17p-deletion.

The scaffold protein NEDD9 is necessary for leukemia-cell migration and disease progression in a mouse model of chronic lymphocytic leukemia

The scaffold protein NEDD9 is frequently upregulated and hyperphosphorylated in cancers, and is associated with poor clinical outcome. NEDD9 promotes B-cell adhesion, migration and chemotaxis, pivotal processes for malignant development. We show that global or B-cell-specific deletion of Nedd9 in chronic lymphocytic leukemia (CLL) mouse models delayed CLL development, markedly reduced disease burden and resulted in significant survival benefit. NEDD9 was required for efficient CLL cell homing, chemotaxis, migration and adhesion. In CLL patients, peripheral NEDD9 expression was associated with adhesion and migration signatures as well as leukocyte count. Additionally, CLL lymph nodes frequently expressed high NEDD9 levels, with a subset of patients showing NEDD9 expression enriched in the CLL proliferation centers. Blocking activity of prominent NEDD9 effectors, including AURKA and HDAC6, effectively reduced CLL cell migration and chemotaxis. Collectively, our study provides evidence for a functional role of NEDD9 in CLL pathogenesis that involves intrinsic defects in adhesion, migration and homing.

Illuminating the molecular mechanisms underlying shoot apical meristem homeostasis in plants.

Unlike animals, terrestrial plants are sessile and able to give rise to new organs throughout their lifetime. In the most extreme cases, they can survive for over a thousand years. With such protracted life cycles, plants have evolved sophisticated strategies to adapt to variable environments by coordinating their morphology as well as their growth, and have consequently acquired a high degree of developmental plasticity, which is supported by small groups of long-lived stem cells found in proliferative centers called meristems. Shoot apical meristems (SAMs) contain multipotent stem cells and provide a microenvironment that ensures both a self-renewable reservoir, to produce primordia and sustain growth, and a differentiating population that develops into all of the above-ground organs of land plants. The homeodomain transcription factor WUSCHEL (WUS) is expressed in the organizing center and acts as a master regulator to govern shoot stem cell homeostasis. In this review, I highlight recent advances in our understanding of the molecular mechanisms and signaling networks that underlie SAM maintenance, and discuss how plants utilize WUS to integrate intrinsic and extrinsic cues.

Multi-objective prediction-based optimization of power consumption for cloud data centers

The overall development of the cloud paradigm is dominating omnipresence in the industry 4.0 business world. Over the last decade, the control measures for power utilization among the proliferative Hyper-Scale Data Centers (HSDCs) have been elucidated. However, the lack of attention to regulating power in Small and Medium-Scale Data Centers (SMSDCs) has ensued in excessive power drainage in small and medium-scale cloud data centers. The crucial factor for excessive power utilization of SMSDCs encompasses providing excessive resources, high certainty tasks. Majority of the previously reported studies zeroed-in on problems associated with hyper-scale data centers, excluding probes of the issues prevalent in small and medium-scale cloud data centers. This paper proffers a framework for a predictive optimization approach for delivering the data center services to end-users. In the first phase, the Multi-Output (MO) Random Forest Regressor (RFR) (MO-RFR) concurrently predicts the multiple-resource utilization of Virtual Machines (VMs). The predictive framework outcome was utilized by the Multi-Objective Particle Swarm Optimization (MO-PSO) framework in the second phase to resolve the issue in virtual machine placement and to accomplish better physical machine consolidation. The proposed multi-prediction-based MO-PSO to escalate the resource usage, minimizes the power utilization, and curtail the carbon footprint. The efficacy of the proposed approach was appraised via performance metrics and actual workload traces. The acquired result from the proposed method outperforms the baseline approaches.