Individual Glomeruli Research Articles

Pheromone representation in the ant antennal lobe changes with age

While the neural basis of age-related decline has been extensively studied,1,2,3 less is known about changes in neural function during the pre-senescent stages of adulthood. Adult neural plasticity is likely a key factor in social insect age polyethism, where individuals perform different tasks as they age and divide labor in an age-dependent manner.4,5,6,7,8,9 Primarily, workers transition from nursing to foraging tasks,5,10 become more aggressive, and more readily display alarm behavior11,12,13,14,15,16 as they get older. While it is unknown how these behavioral dynamics are neurally regulated, they could partially be generated by altered salience of behaviorally relevant stimuli.4,6,7 Here, we investigated how odor coding in the antennal lobe (AL) changes with age in the context of alarm pheromone communication in the clonal raider ant (Ooceraea biroi).17 Similar to other social insects,11,12,16 older ants responded more rapidly to alarm pheromones, the chemical signals for danger. Using whole-AL calcium imaging,18 we then mapped odor representations for five general odorants and two alarm pheromones in young and old ants. Alarm pheromones were represented sparsely at all ages. However, alarm pheromone responses within individual glomeruli changed with age, either increasing or decreasing. Only two glomeruli became sensitized to alarm pheromones with age, while at the same time becoming desensitized to general odorants. Our results suggest that the heightened response to alarm pheromones in older ants occurs via increased sensitivity in these two core glomeruli, illustrating the importance of sensory modulation in social insect division of labor and age-associated behavioral plasticity.

EXPERIMENTAL AND MORPHOLOGICAL JUSTIFICATION OF THE ENTERAL INSUFFICIENCY DEVELOPMENT IN ABDOMINAL SEPSIS

Abstract. Abdominal sepsis continues to remain the most frequent and formidable complication that complicates the course of acute surgical diseases of the abdominal cavity in 15-25 % of cases, and the mortality rate in various forms of peritonitis, depending on the form and its spread, is from 4 % to 60 %, reaching 90 % in postoperative peritonitis.The aim of the study. To study the dynamics of morphological changes of internal organs in animals with simulated acute diff use peritonitis, depending on the stage of the infl ammatory process of the peritoneum.Material and methods. The object of experimental research was 54 white sexually mature non-linear rats weighing 0.18-0.20 kg, kept in the vivarium of the Bukovinian State Medical University, which were divided into two groups. The main group consisted of 28 animals that were simulated with abdominal sepsis. Control group – 26 intact rats. All animals had free access to food and water. Abdominal sepsis in rats was modeled according to our proposed method, which consisted in infecting the peritoneal cavity with the contents of the hollow organs of the abdominal cavity. Animals were removed from the experiment on 1, 3, and 7 days of simulated peritonitis by decapitation under thiopental anesthesia.The results. On the fi rst day of simulation of peritonitis in the animals of the main group, a histological examination of the peritoneum revealed focal purulent peritonitis with damage to the parietal peritoneum. Diff use hyperemia, focal edema of the connective tissue, which is most developed in the submesothelial layers, was observed. Areas of swollen connective tissue contain a slight but diff use mononuclear infl ammatory infi ltrate. We did not detect morphofunctional changes in the liver and kidneys of animals of the main group on the fi rst day of experimental peritonitis. However, already on the third day of the study, the structure of the liver lobe was only partially preserved. The central veins were well visualized, slightly dilated, and contained a small number of erythrocytes. Changes were also found in the lung tissue of animals with peritonitis: enlarged alveoli and congestion of small vessels and hemorrhages. Hemorrhagic syndrome is not excluded as an early manifestation of acute pulmonary insu ffi ciency. On the seventh day, the structure of the liver lobe was signifi cantly disturbed in the animals. The central veins were well visualized, slightly dilated and contained a large number of erythrocytes. Sinusoids were not visualized in the vast majority of fi elds of view, sometimes they were detected only centrilobularly. The beam organization of hepatocytes was disturbed throughout the size of the lobule. During the histological examination of the kidney tissue of the animals on the 7th day of Клінічна анатомія та оперативна хірургія – Т. 22, № 4 – 2023 27 the experiment, we observed that in the cortical layer of the kidney, the glomeruli were somewhat enlarged and dilated, their vessels were fi lled with blood, serous exudate was observed in the lumens, and individual glomeruli were shrunken and collapsed. The vessels of the venous bed were somewhat dilated, full of blood, and small perivascular spot hemorrhages were visualized.Conclusion. Morphological changes occur already during the reactive phase of abdominal sepsis and indicate the stages of multiple organ failure syndrome.

Early Draper-mediated glial refinement of neuropil architecture and synapse number in the Drosophila antennal lobe.

Glial phagocytic activity refines connectivity, though molecular mechanisms regulating this exquisitely sensitive process are incompletely defined. We developed the Drosophila antennal lobe as a model for identifying molecular mechanisms underlying glial refinement of neural circuits in the absence of injury. Antennal lobe organization is stereotyped and characterized by individual glomeruli comprised of unique olfactory receptor neuronal (ORN) populations. The antennal lobe interacts extensively with two glial subtypes: ensheathing glia wrap individual glomeruli, while astrocytes ramify considerably within them. Phagocytic roles for glia in the uninjured antennal lobe are largely unknown. Thus, we tested whether Draper regulates ORN terminal arbor size, shape, or presynaptic content in two representative glomeruli: VC1 and VM7. We find that glial Draper limits the size of individual glomeruli and restrains their presynaptic content. Moreover, glial refinement is apparent in young adults, a period of rapid terminal arbor and synapse growth, indicating that synapse addition and elimination occur simultaneously. Draper has been shown to be expressed in ensheathing glia; unexpectedly, we find it expressed at high levels in late pupal antennal lobe astrocytes. Surprisingly, Draper plays differential roles in ensheathing glia and astrocytes in VC1 and VM7. In VC1, ensheathing glial Draper plays a more significant role in shaping glomerular size and presynaptic content; while in VM7, astrocytic Draper plays the larger role. Together, these data indicate that astrocytes and ensheathing glia employ Draper to refine circuitry in the antennal lobe before the terminal arbors reach their mature form and argue for local heterogeneity of neuron-glia interactions.

89 Bridging Cell Biology and Engineering Sciences: Interdisciplinary Team-based Training in Computational Pathology

OBJECTIVES/GOALS: Computational pathology is an emerging discipline that resides at the intersection of engineering, computer science, and pathology. There is a growing need to develop innovative pedagogical approaches to train future computational pathologists who have diverse educational backgrounds. METHODS/STUDY POPULATION: Our work proposes an iterative approach toward teaching master’s and Ph.D. students from various backgrounds, such as electrical engineering, biomedical engineering, and cell biology the basics of cell-type identification. This approach is grounded in the active learning framework to allow for observation, reflection, and independent application. The learners are trained by a team of an electrical engineer and pathologist and provided with eight images containing a glomerulus. They must then classify nuclei in each of the glomeruli as either a podocyte (blue), endothelial cell (green), or mesangial cell (red). RESULTS/ANTICIPATED RESULTS: A simple web application was built to calculate agreement, measured using Cohen’s kappa, between annotators for both individual glomeruli and across all eight images. Automating the process of providing feedback from an expert renal pathologist to the learner allows for learners to quickly determine where they can improve. After initial training, agreement scores for cells scored by both the learner and the expert were high (0.75), however, when including cells not scored by both the agreement was relatively low (0.45). This indicates that learners needed more instruction on identifying unique cells within each image. This low-stakes approach encourages exploratory and generative learning. DISCUSSION/SIGNIFICANCE: Computation medical sciences require interdisciplinary training methods. We report on a robust approach for team-based mentoring and skill development. Future implementations will include undergraduate learners and provide opportunities for graduate students to engage in near-peer mentoring.

CLINICAL AND PATHOHISTOLOGICAL MANIFESTATIONS OF ACUTE KIDNEY INJURY AMONG PATIENTS WITH ACUTE DRUG POISONING (OVERDOSE).

The aim: To investigate clinical and pathohistological manifestations of acute kidney injury among patients with drug poisoning (overdose). Materials and methods: A cohort retrospective analysis of medical data of 86 patients treated in 2017-2021 with a diagnosis of "acute drug poisoning" com¬plicated by the development of acute kidney injury syndrome was conducted. A forensic medical examination of deceased patients (7 persons) was carried out. Histological samples were examined using an microscope OPTON Axioskop (Germany) in transmitted light, at magnifications of 100 and 400 times. Statistical analysis of the obtained data was carried out using the IBM SPSS Statistics 29.0.0.0 program, Pearson's correlation analysis was used, p≤0.05. Results: Acute renal failure in drug poisoning occurs under the influence of prerenal (hypoxia, r=0,66, р=0,0021; hypovolemia, r=0,61, р=0,0333) and renal factors (toxic effect of chemical components of the drug and rhabdomyolysis, r=0,743, р=0,0034). In the tissue samples, erythrocyte stasis in the capillaries, general fullness of blood vessels, signs of the sludge effect and small diapedesis hemorrhages were found; vasculitis and perivascular sclerosis are noted; foci of mononuclear infiltration of the stroma, focal edema, necrosis and interstitial fibrosis; desquamation, degenerative-dystrophic changes of the nephrothelium, tubular atrophy were found; hyaline casts in separate tubules; focal glomerular changes with segmental increase of the mesangial matrix and proliferation of endothelial cells, atrophy and hyalinosis of individual glomeruli were noted. Conclusions: The multifactorial effect of opioids is confirmed by microcirculation disorders, vascular, interstitial, tubular and glomerular changes in the kidneys.

Digital Spatial Profiling of Glomerular Gene Expression in Pauci-Immune Focal Necrotizing Glomerulonephritis.

Pauci-immune focal necrotizing glomerulonephritis (piFNGN) involves asynchronous onset and progression of injurious lesions in biopsies. Pathologists can describe this heterogeneity within a biopsy, but translating the information into prognostic or expression analyses is challenging. Understanding the underlying molecular processes could improve treatment; however, bulk or single-cell transcriptomic analyses of dissociated tissue disregard the heterogeneity of glomerular injury. We characterize protein and mRNA expression of individual glomeruli in 20 biopsies from 18 patients with antineutrophil cytoplasmic antibody-associated piFNGN using the NanoString digital spatial profiling (DSP) platform. For this purpose, circular annotations of glomeruli were analyzed using protein, immuno-oncology RNA, and Cancer Transcriptome Atlas panels (n=120, 72, and 48 glomeruli, respectively). Histologic evaluation of glomerular patterns of injury was performed in adjacent serial sections. Expression data were processed by log2 transformation, quantile normalization, and batch adjustment. DSP revealed distinct but overlapping gene expression profiles relating to the morphological evolution of injurious lesions, including dynamic expression of various immune checkpoint regulators. Enrichment analysis indicated deregulated pathways that underline known and highlight novel potential mechanisms of disease. Moreover, by capturing individual glomeruli, DSP describes heterogeneity between and within biopsies. We demonstrate the benefit of spatial profiling for characterization of heterogeneous glomerular injury, indicating novel molecular correlates of glomerular injury in piFNGN.

Digital spatial profiling of glomerular gene expression in pauci-immune focal necrotizing glomerulonephritis

Introduction: Pauci-immune focal necrotizing glomerulonephritis (piFNGN) involves asynchronous onset and progression of injurious lesions in biopsies. Pathologists describe this heterogeneity within a biopsy, but translating the information into prognostic or expression analyses is challenging. Understanding the underlying molecular processes could improve treatment; however, bulk or single cell transcriptomic analyses of dissociated tissue disregard the heterogeneity of glomerular injury. Methods: We characterize protein and mRNA expression of individual glomeruli in 20 biopsies from 18 patients with anti-neutrophil cytoplasmic antibody-associated piFNGN using the Nanostring digital spatial profiling (DSP) platform. For this purpose, circular annotations of glomeruli were analyzed using protein, immuno-oncology RNA, and cancer transcriptome atlas panels (n=120, 72, and 48 glomeruli, respectively). Histological evaluation of glomerular patterns of injury was performed in adjacent serial sections. Expression data was processed by log2 transformation, quantile normalization and batch adjustment. Results: DSP revealed distinct but overlapping gene expression profiles relating to the morphological evolution of injurious lesions, including dynamic expression of various immune checkpoint regulators. Enrichment analysis indicated deregulated pathways that underline known and highlight novel potential mechanisms of disease. Moreover, by capturing individual glomeruli, DSP describes heterogeneity between and within biopsies. Conclusions: We demonstrate the benefit of spatial profiling for characterization of heterogeneous glomerular injury, indicating novel molecular correlates of glomerular injury in piFNGN.

Ada-CCFNet: Classification of multimodal direct immunofluorescence images for membranous nephropathy via adaptive weighted confidence calibration fusion network

In the pathological diagnosis of early, late and non-membranous nephropathy, direct immunofluorescence is highly likely to present potentially specific lesions, while it is often overlooked due to the difficulty of screening with naked eyes. With the advanced progress of deep learning, they have shown powerful abilities in detecting potential lesions. In this paper, we propose an adaptive weighted confidence calibration fusion framework (Ada-CCFNet) consisting of a preprocessing module, an adaptive weighted confidence calibration fusion (Ada-CCF) module and a classification module for diagnosis of membranous nephropathy by classifying the multimodal direct immunofluorescence images. In the preprocessing module, we use the well-known U-Net to segment individual glomeruli and standardize their luminance appearance by the average luminance difference method, allowing the subsequent modules to focus more on the diseased glomerular region. Subsequently, in the Ada-CCF module, six confidence calibration methods are utilized for two main direct immunofluorescence images, IgG and C3, and the comprehensive calibration scores are obtained based on the adaptive weighted fusion of six confidence calibration methods to obtain more reliable confidence level, in which the adaptive weights are related with expected calibration error reductions. For the classification module, the weighted probability scores of IgG and C3 are jointly fed into the module to achieve the classification by random forest. Experimental results showed that Ada-CCFNet achieves the classification accuracy of 73.52%, surpassing the methods of using single IgG or C3 images and positive grade indicator with 8.24%, 8.94% and 22.76%, and outperforming the compared methods in the classification of membranous nephropathy.

A quantitative 3D intravital look at the juxtaglomerular renin-cell-niche reveals an individual intra/extraglomerular feedback system.

The juxtaglomerular niche occupied by renin cells (RCN) plays an important role in glomerular repair but the precise temporal and spatial interrelations remain unclear. This study proposes the hypothesis of a local intra-extraglomerular regenerative feedback system and establishes a new quantifiable system for RCN responses in individual glomeruli in vivo. A strictly intraglomerular two-photon laser-induced injury model was established. Labeled renin cells (RC) in transgenic renin reporter mice were fate-traced in healthy and injured glomeruli over several days by intravital microscopy and quantified via new three-dimensional image processing algorithms based on ray tracing. RC in healthy glomeruli demonstrated dynamic extraglomerular protrusions. Upon intraglomerular injury the corresponding RCN first increased in volume and then increased in area of dynamic migration up to threefold compared to their RCN. RC started migration reaching the site of injury within 3 hours and acquired a mesangial cell phenotype without losing physical RCN-contact. During intraglomerular repair only the corresponding RCN responded via stimulated neogenesis, a process of de novo differentiation of RC to replenish the RCN. Repeated continuous intravital microscopy provides a state-of-the-art tool to prove and further study the local intraglomerular RCN repair feedback system in individual glomeruli in vivo in a quantifiable manner.

Dense Multi-Object 3D Glomerular Reconstruction and Quantification on 2D Serial Section Whole Slide Images.

There has been a long pursuit for precise and reproducible glomerular quantification in the field of renal pathology in both research and clinical practice. Currently, 3D glomerular identification and reconstruction of large-scale glomeruli are labor-intensive tasks, and time-consuming by manual analysis on whole slide imaging (WSI) in 2D serial sectioning representation. The accuracy of serial section analysis is also limited in the 2D serial context. Moreover, there are no approaches to present 3D glomerular visualization for human examination (volume calculation, 3D phenotype analysis, etc.). In this paper, we introduce an end-to-end holistic deep-learning-based method that achieves automatic detection, segmentation and multi-object tracking (MOT) of individual glomeruli with large-scale glomerular-registered assessment in a 3D context on WSIs. The high-resolution WSIs are the inputs, while the outputs are the 3D glomerular reconstruction and volume estimation. This pipeline achieves 81.8 in IDF1 and 69.1 in MOTA as MOT performance, while the proposed volume estimation achieves 0.84 Spearman correlation coefficient with manual annotation. The end-to-end MAP3D+ pipeline provides an approach for extensive 3D glomerular reconstruction and volume quantification from 2D serial section WSIs.

Digital Spatial Profiling of Individual Glomeruli From Patients With Anti-Neutrophil Cytoplasmic Autoantibody-Associated Glomerulonephritis.

We previously showed that the rupture of Bowman’s capsule (BC) promotes the progression of crescentic glomerulonephritis by enhancing the entry of CD8+ T cells into the glomeruli. In the present study, we utilized digital spatial profiling to simultaneously profile the altered abundances of the messenger RNA (mRNA) transcripts and proteins in the glomerular and periglomerular areas of four biopsy samples of anti-neutrophil cytoplasmic autoantibody-associated glomerulonephritis (ANCA-GN) and two biopsy specimens of minimal change disease (MCD) controls. The paraffin-embedded biopsy samples were stained with collagen IV, CD45, and SYTO 13 to distinguish the glomeruli with periglomerular infiltration but intact BC, with focal BC rupture, and with extensive rupture of BC and glomeruli without crescent formation and leukocytic infiltration in ANCA-GN. By assessing multiple discrete glomerular areas, we found that the transcript expression levels of the secreted phosphoprotein-1 and its receptor CD44 were upregulated significantly in the glomeruli with more severe ruptures of BC, and their expression levels correlated positively with the fibrotic markers. We also found that both alternative and classic complement pathways were activated in the glomeruli from patients with ANCA-GN. Furthermore, M1 macrophages were involved mostly in the early stage of BC rupture, while M2 macrophages were involved in the late stage and may contribute to the fibrosis process of the crescents. Finally, loss of glomerular cells in ANCA-GN was likely mediated by apoptosis. Our results show that digital spatial profiling allows the comparative analysis of the mRNA and protein profiles in individual glomeruli affected differently by the disease process and the identification of potential novel mechanisms in ANCA-GN.

Digital spatial profiling of collapsing glomerulopathy

Collapsing glomerulopathy is a histologically distinct variant of focal and segmental glomerulosclerosis that presents with heavy proteinuria and portends a poor prognosis. Collapsing glomerulopathy can be triggered by viral infections such as HIV or SARS-CoV-2. Transcriptional profiling of collapsing glomerulopathy lesions is difficult since only a few glomeruli may exhibit this histology within a kidney biopsy and the mechanisms driving this heterogeneity are unknown. Therefore, we used recently developed digital spatial profiling (DSP) technology which permits quantification of mRNA at the level of individual glomeruli. Using DSP, we profiled 1,852 transcripts in glomeruli isolated from formalin fixed paraffin embedded sections from HIV or SARS-CoV-2-infected patients with biopsy-confirmed collapsing glomerulopathy and used normal biopsy sections as controls. Even though glomeruli with collapsing features appeared histologically similar across both groups of patients by light microscopy, the increased resolution of DSP uncovered intra- and inter-patient heterogeneity in glomerular transcriptional profiles that were missed in early laser capture microdissection studies of pooled glomeruli. Focused validation using immunohistochemistry and RNA in situ hybridization showed good concordance with DSP results. Thus, DSP represents a powerful method to dissect transcriptional programs of pathologically discernible kidney lesions.

Parallel Processing of Olfactory and Mechanosensory Information in the Honey Bee Antennal Lobe.

In insects, neuronal responses to clean air have so far been reported only episodically in moths. Here we present results obtained by fast two-photon calcium imaging in the honey bee Apis mellifera, indicating a substantial involvement of the antennal lobe, the first olfactory neuropil, in the processing of mechanical stimuli. Clean air pulses generate a complex pattern of glomerular activation that provides a code for stimulus intensity and dynamics with a similar level of stereotypy as observed for the olfactory code. Overlapping the air pulses with odor stimuli reveals a superposition of mechanosensory and odor response codes with high contrast. On the mechanosensitive signal, modulations were observed in the same frequency regime as the oscillatory motion of the antennae, suggesting a possible way to detect odorless airflow directions. The transduction of mechanosensory information via the insect antennae has so far been attributed primarily to Johnston’s organ in the pedicel of the antenna. The possibility that the antennal lobe activation by clean air originates from Johnston’s organ could be ruled out, as the signal is suppressed by covering the surfaces of the otherwise freely moving and bending antennae, which should leave Johnston’s organ unaffected. The tuning curves of individual glomeruli indicate increased sensitivity at low-frequency mechanical oscillations as produced by the abdominal motion in waggle dance communication, suggesting a further potential function of this mechanosensory code. The discovery that the olfactory system can sense both odors and mechanical stimuli has recently been made also in mammals. The results presented here give hope that studies on insects can make a fundamental contribution to the cross-taxa understanding of this dual function, as only a few thousand neurons are involved in their brains, all of which are accessible by in vivo optical imaging.

Glomerular Size in Transplanted Kidneys: Does Size Matter?

Glomerular Size in Transplanted Kidneys: Does Size Matter?

Image analysis techniques to map pyramids, pyramid structure, glomerular distribution, and pathology in the intact human kidney from 3-D MRI.

Kidney pathologies are often highly heterogeneous. To comprehensively understand kidney structure and pathology, it is critical to develop tools to map tissue microstructure in the context of the whole, intact organ. Magnetic resonance imaging (MRI) can provide a unique, three-dimensional view of the kidney and allows for measurements of multiple pathological features. Here, we developed a platform to systematically render and map gross and microstructural features of the human kidney based on three-dimensional MRI. These features include pyramid number and morphology as well as the associated medulla and cortex. In a subset of these kidneys, we also mapped individual glomeruli and glomerular volumes using cationic ferritin-enhanced MRI to report intrarenal heterogeneity in glomerular density and size. Finally, we rendered and measured regions of nephron loss due to pathology and individual glomerular volumes in each pyramidal unit. This work provides new tools to comprehensively evaluate the kidney across scales, with potential applications in anatomic and physiological research, transplant allograft evaluation, biomarker development, biopsy guidance, and therapeutic monitoring. These image rendering and analysis tools could eventually impact the field of transplantation medicine to improve longevity matching of donor allografts and recipients and reduce discard rates through the direct assessment of donor kidneys.NEW & NOTEWORTHY We report the application of cutting-edge image analysis approaches to characterize the pyramidal geometry, glomerular microstructure, and heterogeneity of the whole human kidney imaged using MRI. This work establishes a framework to improve the detection of microstructural pathology to potentially facilitate disease monitoring or transplant evaluation in the individual kidney.

Endothelial Endothelin Receptor A Expression Is Associated With Podocyte Injury and Oxidative Stress in Patients With Focal Segmental Glomerulosclerosis

IntroductionThe podocyte is thought to be the mainly affected cell type in focal segmental glomerulosclerosis (FSGS). However, recent studies have also indicated a role for glomerular endothelial cells and podocyte−endothelial crosstalk in FSGS development. An experimental model for podocyte injury showed that increased endothelin-1 (ET-1) signaling between podocytes and endothelial cells induces endothelial oxidative stress and subsequent podocyte loss. In the current study, we investigated endothelial endothelin receptor A (ETAR) expression in patients with FSGS and its association with podocyte injury and glomerular oxidative stress.MethodsWe selected 39 biopsy samples of patients with FSGS and 8 healthy control subjects, and stained them for ETAR, nephrin and 8-oxo-guanine, a DNA lesion caused by oxidative damage. Glomeruli with ETAR-positive endothelium and with nephrin loss were scored, and the 8-oxo-guanine−positive glomerular area was measured.ResultsThe mean percentage of glomeruli with ETAR-positive endothelial cells in patients with FSGS was higher compared to that in healthy control subjects (52% vs. 7%; P < 0.001). The presence of glomerular ETAR-positive endothelium was strongly associated with nephrin loss both on the biopsy level (rho = 0.47; P < 0.01), as on the level of individual glomeruli (odds ratio = 2.0; P < 0.001). Moreover, glomeruli with ETAR-positive endothelium showed more 8-oxo-guanine−positive staining (1.9% vs. 2.4%; P = 0.037). Finally, 8-oxo-guanine positivity in glomeruli was associated with increased levels of proteinuria.ConclusionTaking together our findings, we show that ETAR is increased in glomerular endothelial cells of patients with FSGS and associated with podocyte damage and glomerular oxidative stress. These findings support the hypothesis that ET-1 signaling in glomerular endothelial cells contributes to disease development in patients with FSGS.

Neurodevelopmental and transcriptomic effects of CRISPR/Cas9-induced somatic orco mutation in honey bees

In insects, odorant receptors facilitate olfactory communication and require the functionality of the highly conserved co-receptor gene orco. Genome editing studies in a few species of ants and moths have revealed that orco can also have a neurodevelopmental function, in addition to its canonical role in adult olfaction, discovered first in Drosophila melanogaster. To extend this analysis, we determined whether orco mutations also affect the development of the adult brain of the honey bee Apis mellifera, an important model system for social behavior and chemical communication. We used CRISPR/Cas9 to knock out orco and examined anatomical and molecular consequences. To increase efficiency, we coupled embryo microinjection with a laboratory egg collection and in vitro rearing system. This new workflow advances genomic engineering technologies in honey bees by overcoming restrictions associated with field studies. We used Sanger sequencing to quickly select individuals with complete orco knockout for neuroanatomical analyses and later validated and described the mutations with amplicon sequencing. Mutant bees had significantly fewer glomeruli, smaller total volume of all the glomeruli, and higher mean individual glomerulus volume in the antennal lobe compared to wild-type controls. RNA-Sequencing revealed that orco knockout also caused differential expression of hundreds of genes in the antenna, including genes related to neural development and genes encoding odorant receptors. The expression of other types of chemoreceptor genes was generally unaffected, reflecting specificity of CRISPR activity in this study. These results suggest that neurodevelopmental effects of orco are related to specific insect life histories.

Structural Features of Neuro-Glio-Vascular Ensembles in the Glomeruli of the Rat Olfactory Bulb

The glomeruli of the olfactory bulb of mammals are the primary coding elements of olfactory information. The excitation pattern produced by individual glomeruli in response to olfactory stimulation is stable and specific to certain odors. First of all, this is due to the structure of the neural circuits of the olfactory bulb. Nevertheless, there is reason to believe that auxiliary cells of the nervous system—astrocytes—play a role in the organization of the primary processing of the olfactory signal. It is known that astrocytes not only have a direct effect on synaptic activity and plasticity, but coordinate the joint work of neuronal circuits and the vascular component, forming so-called “neuro-glio-vascular ensembles” as well. In this study, we carried out a morphological study of peripheral processes and gap junctions of astrocytes in order to study the structure of neuro-glio-vascular ensembles at the level of organization of the olfactory bulb glomeruli neuropil. The study showed that the main part of the astrocytic processes inside the glomeruli is located in the area of dendro-dendritic connections of the interneurons and projection neurons of the olfactory bulb, while in the zone of the primary switching of the olfactory signal, the astrocytic processes are practically absent. We also found a pronounced imbalance in the expression of the main astroglial connexins between the different functional poles of the neuro-glio-vascular ensembles of the olfactory bulb and the presence of heterotypic contacts formed by Cx30. The functional significance of the observed features of connexins expression in the olfactory bulb has yet to be studied.

Detection of Mesangial hypercellularity of MEST-C score in immunoglobulin A-nephropathy using deep convolutional neural network

Immunoglobulin A (IgA)-nephropathy (IgAN) is one of the major reasons for renal failure. It provides vital clues to estimate the stage and the proliferation rate of end-stage kidney disease. IgA stage can be estimated with the help of MEST-C score. The manual estimation of MEST-C score from whole slide kidney images is a very tedious and difficult task. This study uses some Convolutional neural networks (CNNs) related models to detect mesangial hypercellularity (M score) in MEST-C. CNN learns the features directly from image data without the requirement of analytical data. CNN is trained efficiently when image data size is large enough for a particular class. In the case of smaller data size, transfer learning can be used efficiently in which CNN is pre-trained on some general images and then on subject images. Since the data set size is small, time spent in collecting large data set is saved. The training time of transfer learning is also reduced because the model is already pre-trained. This research work aims at the detection of mesangial hypercellularity from biopsy images with small data size by utilizing the transfer learning. The dataset used in this research work consists of 138 individual glomerulus (× 20 magnification digital biopsy) images of IgA patients received from All India Institute of Medical Science, Delhi. Here, machine learning (k-nearest neighbour (KNN) and support vector machine (SVM)) classifiers are compared to transfer learning CNN methods. The deep extracted image features are used by machine learning classifiers. The different evaluation parameters have been used for comparing the predictions of basic classifiers to the deep learning model. The research work concludes that the transfer learning deep CNN method can improve the detection of mesangial hypercellularity as compare to KNN, SVM methods when using the small data set. This model could help the pathologists to understand the stages of kidney failure.

Probabilistic modeling of Diabetic Nephropathy progression.

Diabetic Nephropathy (DN) progression is stratified into several stages with different levels of proteinuria, albuminuria, and physical characteristics as observed by pathologists. These physical changes are primarily visible within a patient's glomeruli which function as filtration units for blood returning for oxygenation. As DN stage increases, it is possible to observe the thickening of the glomerular basement membrane, expansion of the mesangium, and development of nodular sclerosis. Classification of different stages of DN by pathologists is based on semi-qualitative assessments of these characteristics on an individual glomerulus basis. Being able to probabilistically infer stage membership of individual glomeruli based on a combination of easily observable and hidden image features would be an invaluable tool for furthering our understanding of the drivers of DN progression. Markov Particle filters, included in the bnlearn package in R, were used to query a Bayesian Network (BN) constructed using the structural Hill-Climbing algorithm on a set of glomerular features. These features included both traditional characteristics such as glomerular area and number of mesangial nuclei as well as more abstract features derived from Minimum Spanning Trees (MST) to quantify spatial distribution of mesangial nuclei. Our results using images from multiple institutions suggest that these abstract features exercise a variable influence on DN stage membership over the course of disease progression. Further research incorporating clinical data will give nephrologists a "white box" visual of quantitative factors present in DN patients.