Smoother Research Articles

Stereological study of organelle distribution in human mature oocytes

The ultrastructure of human oocytes has been described only qualitatively. To offer a precise organelle spatial distribution and organelle volume during the main maturation stages, we previously conducted stereological studies on prophase-I (GV) and metaphase-I (MI) oocytes, and here we present results on metaphase-II (MII) oocytes. Five donor oocytes from different donors were processed for transmission electron microscopy, and quantification of organelle distribution was performed using point-counting stereology. Statistical tests compared the means of the relative volumes occupied by organelles among oocyte regions. The most abundant organelles were elements of the smooth endoplasmic reticulum (SER), such as SER small vesicles, SER medium vesicles, SER large vesicles and SER isolated tubules, along with mitochondria, followed by SER tubular aggregates, cortical vesicles and lysosomes. Significant differences between oocyte regions were found for lysosomes, cortical vesicles and SER large vesicles. Comparisons of MII oocytes to previous findings in GV and MI oocytes evidenced specific patterns of organelle distribution and relative volumes. This final evaluation thus enables to track organelle spatial reorganization across oocyte stages, which, in addition to gathered knowledge, may be useful to assist in improvements of stimulation protocols, in-vitro maturation media and cryopreservation techniques.

Blastocysts originated from oocytes with smooth endoplasmic reticulum aggregates have a reduced euploidy rate: a retrospective cohort study.

Does the presence of smooth endoplasmic reticulum aggregates (SERa) in oocytes adversely impact the euploidy rate of subsequent blastocysts? We performed a retrospective cohort study with 671 young patients (< 38 years) undergoing their first preimplantation genetic testing for aneuploidy (PGT-A) between January 2019 and October 2022 at a reproductive medical center of university affiliated teaching hospitals in China. Cycles were categorized as either SERa(+) cycles (containing at least one SERa(+) oocyte) or SERa(-) cycles (all oocytes without SERa). In SERa(+) cycles, oocytes were further subdivided into the SERa(+) oocyte group and the sibling SERa(-) oocyte group, comprising oocytes with normal morphology. No significant differences were observed in the normal fertilization rate (72.9% vs. 75.4% vs. 72.6%, P=0.343), and cleavage rate (96.8% vs. 97.1% vs. 96.4%, P=0.839) among the SERa(-) cycle group, the SERa(-) oocyte group, and the SERa(+) oocyte group. Additionally, there were no statistically significant differences in the rates of good quality embryos (44.7% vs. 48.8% vs. 46.2%, P=0.177) or blastocyst formation (60.1% vs. 60.9% vs. 60.5%, P=0.893) among the groups. However, the euploidy rate of blastocysts derived from SERa(+) oocytes was significantly lower compared to those from SERa(-) oocytes in SERa(+) cycles and normal oocytes in SERa(-) cycles (39.3% vs. 51.2% vs. 54.5%, P=0.005). Despite this, there were no significant differences in pregnancy and neonatal outcomes after euploid embryo transfer among the three groups. Blastocysts derived from SERa(+) oocytes have a lower euploidy rate than those derived from SERa(-) oocytes. Nevertheless, comparable reproductive outcomes were achieved following euploid embryo transfer from both SERa(+) and SERa(-) oocytes.

Remodeling of the structural components of the capsule and glomerular zone of the adrenal glands cortex of white rats under the influence of a complex of food additives at the late terms of the experimental study

With food additives, we can add flavour to food, extend its shelf life, and make it look good. However, even small amounts of these additives can lead to diseases of various organs and systems. The aim of our work is to establish metrics and morphological changes in the structure of the capsule and the glomerular zone of the cortex of the adrenal glands of rats at the later stages of the experimental study under the conditions of consumption of a complex of food additives: monosodium glutamate, sodium nitrite and Ponceau 4R. The control group of rats consumed oral drinking water and saline. The rats of the experimental groups were orally administered once a day with a 10 % solution of sodium nitrite (E250), sodium glutamate (E621) or Ponceau 4R. The dosages of food additives were two times lower than the permissible norm in food. The rats were withdrawn from the experiment after 8, 12 and 16 weeks. The adrenal glands were removed for the study. Paraffin sections were stained with hematoxylin and eosin, and semi-thin sections with toluidine blue, and examined using a light microscope Biorex with a digital microphoto attachment DSM 900. Ultrathin sections were examined in a TEM-125 K electron microscope. For morphometric analysis, the structure of the capsule and the glomerular zone of the cortex of the adrenal glands of rats at the later stages of the experimental study was studied. The study results were analysed using the software package “InStat”. At the late stages of the experimental study, the adrenal capsule underwent irreversible changes. The average thickness of the adrenal connective tissue capsule decreased by the end of the 16th week of the experimental research. Similarly, the average thickness of the cortex decreased with each subsequent week relative to the control values, indicating significant structural and functional disorders. The average thickness of the glomerular zone of the adrenal cortex periodically changed as a compensatory reaction in response to the administration of a complex of food additives to rats. The phenomena of nucleus pyknosis and sludge syndrome were detected, which indicated compensatory and adaptive processes at the microscopic level. The analysis of electron micrographs revealed destroyed granular endoplasmic reticulum cisternae and mitochondrial remnants. Thus, it has been established that using a complex of food additives (monosodium glutamate, sodium nitrite and Ponceau 4R) directly affects the adrenal glands of rats. This effect causes structural changes in the cortex and medulla, causing alteration and hypertrophy of cells in these areas. Additionally, hyperhydration of the connective tissue stroma and compensatory-restorative reactions aimed at transcription factors and internal mechanisms of nitric oxide formation were recorded.

Morphological and cytochemical characterization of the peripheral blood cells in Paralichthys olivaceus

The flounder (Paralichthys olivaceus) is a highly nutritious, cultured bony fish with a high economic value. The health of the fish is closely related to its blood cells, which are critical for oxygen transport, natural defense, and immunity. The microstructures, classification, counting and size of peripheral blood cells in P. olivaceus were observed and measured by Wright-Giemsa staining, and the cytochemical characteristics of peripheral blood cells were investigated by different cytochemical staining methods including periodic acid-Schiff (PAS), Sudan black B (SBB), acid phosphatase (ACP), alkaline phosphatase (ALP), peroxidase (POX), and α-naphthol acetate esterase (NAE). Besides, the ultrastructures of different cells were detected by the transmission electron microscope. The results showed that erythrocytes, thrombocytes, lymphocytes, monocytes, and neutrophils constituted the peripheral blood cells in P. olivaceus. More heterochromatins were found in erythrocytes, thrombocytes and neutrophils; however, more organelles with fewer heterochromatins were found in monocytes. Endoplasmic reticulums and phagocytic vesicles were abundant in neutrophils. Lymphocytes were the most abundant in leukocytes, followed by monocytes and neutrophils. The cytochemical staining results showed that all leukocytes were positive for SBB. Most of the lymphocytes were positive for PAS, and monocytes were positive for PAS, ACP, and POX. As for neutrophils, ACP and POX were positive. Both monocytes and neutrophils showed positive for SBB, ACP and POX, indicating that the two kinds of cells play a vital role in phagocytosis and bactericidal action. Only lymphocytes were positive for ALP, indicating that they were important in inflammation and immune response. Some characteristics similarities in peripheral blood cells were showed in P. olivaceus just as the other fishes.

FEATURES OF STRUCTURAL REORGANISATION OF THE HYPOTHALAMIC SUPRAOPTIC NUCLEUS IN RATS DURING THE EARLY STAGES OF DIABETES DEVELOPMENT

The objective of the study – to ascertain the structural organisation of the hypothalamicsupraoptic nucleus in rats with streptozotocin- induced diabetes.Materials and Methods. Mature rats, modelled with streptozotocin- induced diabetes,were used in the experiment. On the 14th day of the experimental diabetes developmentin rats, quantitative cyto-myeloarchitecture parameters, histological and ultrastructuralchanges in the anterior hypothalamic supraoptic nucleus were determined.Results. After 14 days of the experiment modelling, the histological structure of thehypothalamic supraoptic nucleus remains practically unchanged and identical to the intactanimals. However, occasional occurrences of central chromatolysis were observed in thelight neurons of the supraoptic nucleus. The perikarya of the dark neurons were denselyfilled with Nissl substance. The ultrastructural feature of the dark neurosecretory cellsis the presence of highly developed granular endoplasmic reticulum in their peripheralzone, which was represented by flat elongated cisterns. These cisterns had a large numberof ribosomes on their surface. Additionally, free ribosomes and polysomes were present inthe hyaloplasm between the cisterns. A similar pattern has been observed in light neurons.Furthermore, an increase in the volumetric density of neurosecretory granules comparedto intact indicators of up to 2.36 ± 0.12 % (p ≤ 0.01) has been detected. In the neuropil of the hypothalamus, most of the axons were filled with neurosecretory granules (NG)of moderate electron density. Alongside axons that were expanded and filled withneurosecretory granules, axons of neurosecretory cells with usual structure containingmitochondria, synaptic vesicles, neurotubules, and neurofilaments have been observed.Conclusions. 1. The hypothalamic supraoptic nucleus contained light and darkneurosecretory cells with a well-developed protein synthesis apparatus. 2. Ultrastructuralchanges of the neuro-glio-capillary complexes in the investigated hypothalamic nuclei weredetected on the 14th day of the experimental streptozotocin- induced diabetes, indicatingthe activation of neurosecretion synthesis and the transport to the neurohypophysis. Anincrease in the volumetric density of neurosecretory granules compared to intact animalswas found to be 2.36±0.12 % (p≤0.01).

The ultrastructure of peroxisomes in the kidney of the camel (Camelus dromedarius).

Dromedary camels can survive and reproduce in desert areas. The unique anatomical structure of the kidney enables the camel to prevent water loss. The present study aimed to investigate the ultrastructure of the peroxisomes in the normal kidney of the adult dromedary camel. Tissue samples were taken from the cortex and outer medulla of the kidney of eight camels. The samples were then processed for histological and ultrastructural investigations. The epithelial cells of the proximal tubules displayed peroxisomes with varying sizes and shapes. The peroxisomes were observed in either dispersed or clustered arrangement. Each peroxisome exhibited a homogenous matrix enveloped by a single membrane. Several peroxisomes exhibited one or more dark marginal plates that were always strongly associated with the smooth endoplasmic reticulum. The intensity of the peroxisomal matrix differed significantly, either within the same cell or across different cells. The intensity was light or dark, with a few peroxisomes presenting a similar intensity to that of the mitochondria. Some peroxisomes contained nucleoids within their matrix. The peroxisomes in the first and second sections of proximal convoluted tubules were scattered and primarily located in the region between the microvilli and the underlying mitochondria. The peroxisomes in the third region were abundant and frequently aggregated in clusters throughout the cytoplasm. In the fourth region, the number of peroxisomes was low. The proximal straight tubule had a limited quantity of peroxisomes. In conclusion, peroxisomes in the proximal tubule in kidney of normal dromedary camel were similar in shape and size to other mammals; however, heterogeneity exists as a result of differences in species-specific peroxisomal proteins. Peroxisomes are suggested to be a major source of metabolic energy and act as hydrogen peroxide (H2O2) scavengers, resulting in the release of water and oxygen.

Morphological and histological observations on the hair brush of Endoclita vietnamensis (Lepidoptera, Hepialidae).

The most prominent insect pest harming eucalyptus trees in China was Endoclita vietnamensis, which resulted in considerable losses to the environment and economy. This study examined the hair brush on the postpedes of male E. vietnamensis using transmission and scanning electron microscopy. The finding revealed that the hair brush was made up of many large, flattened, pale yellow, specialized scales that were highly elongated. These scales were firmly affixed to the epidermis at their base, and at the attachment site, there was a swollen hair follicle. The interior of the pencil was hollow with ducts, and the surface featured rows of pores and longitudinal ridges. Numerous features, including mitochondria, lipid droplets, smooth and rough endoplasmic reticulum, and microvilli, were present in the tissue cells of the tibial segment of postpedes of male moths. The tibial segment of postpedes of male moths exhibited thicker epidermis, cavities, and voids in the tissue cortex, suggesting the presence of secretory activities in the tissue cells. With the location and structure of the pheromone gland determined, a theoretical foundation for future research into the communication between the female and male E. vietnamensis as well as the development of sex-specific pheromone-based catching and killing methods will be available. RESEARCH HIGHLIGHTS: Endoclita vietnamensis Buchsbaum & Grehan, 2022 is the most prominent insect pest harming eucalyptus trees in China. With the location and structure of the pheromone gland determined, a theoretical foundation for future research into the communication between the female and male E. vietnamensis as well as the development of sex-specific pheromone-based catching and killing methods will be available.

Smooth endoplasmic reticulum aggregates in oocytes associated with increased risk of neonatal birth defects: Ameta-analysis.

Previous studies have indicated the association between smooth endoplasmic reticulum aggregates (SERa+) and poorer medically assisted reproduction outcomes. However, the link between SERa+ and neonatal outcomes remains controversial and open for debate. A comprehensive meta-analysis on the relation between SERa+ and the risk of birth defects is needed. The literature search was conducted using the following databases: PubMed, Embase, Cochrane Libraries, Web of Science, and Chinese databases including China National Knowledge Infrastructure (CNKI) and Wan Fang from inception until July 2023. Risk ratio (RR) and 95% confidence interval (CI) were calculated by a fixed-effected model, while heterogeneity was assessed by forest plots and I2 statistic. Funnel plot was produced to assess publication bias. This meta-analysis has been registered on PROSPERO (CRD42022313387). The search resulted in 122 studies, 14 of which met the inclusion criteria. The analysis of birth defects revealed a higher risk (RR = 2.17, 95%CI 1.24 to 3.81, p = 0.007) in children derived from SERa+ cycle compared to SERa- cycles (711 vs. 4633). Meanwhile, in a subgroup analysis, the risk of birth defects was significantly increased in the SERa+ oocytes group as compared with the sibling SERa- oocytes group (RR = 3.53, 95%CI 1.21 to 10.24, p = 0.02). To conclude, our analysis indicated that SERa+ cycles/oocytes may have a potential risk of increased additional major birth defects comparing with SERa- cycles/oocytes. This conclusion may provide evidence-based support for clinicians in IVF clinical guidance and embryologists in prudent embryo selection strategy.

P-132 Outcomes of blastocyst transfer derived from oocytes with smooth endoplasmic reticulum clusters (sERC)

Abstract Study question Are blastocysts derived from oocytes with smooth endoplasmic reticulum clusters (sERC+) as suitable for transfer as those from oocytes without such clusters (sERC-)? Summary answer Blastocysts from sERC+ oocytes are considered for embryo transfer; however, it is recommended to prioritize them lower than those from sERC- oocytes. What is known already One of the cytoplasmic abnormalities in human oocytes, sERC, typically manifests during the MII stage oocytes and disappears after fertilization when the female and male pronuclei emerge. Despite this, the mechanism behind sERC development remains unclear. While it is suggested that sERC in oocytes may adversely impact embryo development and implantation, a definitive conclusion on its safety or use has not been reached. Although the 2011 Istanbul consensus discouraged injecting or inseminating sERC+ oocytes, a 2017 expert panel reconsidered this recommendation, advocating for a case-by-case approach. Therefore, there are widespread variations in responses to sERC in clinical practices. Study design, size, duration A retrospective review of intracytoplasmic sperm injection (ICSI) cycles between April 2014 and April 2023 was conducted, exclusively on single frozen blastocyst transfers of two pronuclei (2PN)-derived embryos performed before June 2023. A dataset comprising 95 blastocysts from sERC+ oocytes and 8,698 blastocysts from sERC- oocytes was assembled. To reduce bias between groups, maternal age at retrieval and transfer counts were matched in a 1:1 ratio using propensity score matching, yielding 95 comparable cycle pairs. Participants/materials, setting, methods Among all ICSI cycles, 78 exclusively involved embryos from sERC+ oocytes, 847 included embryos from both sERC+ and sERC- oocytes, and 6,599 had no embryos derived from sERC+ oocytes. Among the 8,698 sERC- blastocysts transferred, 1,083 and 7,615 originated from cycles with and without sERC+ oocytes, respectively. Implantation, miscarriage, and live birth rates were then compared in 95 matched pairs of blastocysts derived from sERC+ and sERC- oocytes. Main results and the role of chance Following propensity score matching adjustments, the mean patient age at retrieval (mean ± SD) for blastocyst transfers derived from sERC+ and sERC- oocytes were 38.6 ± 4.7 years and 38.7 ± 4.8 years, respectively (p = 0.927). The transfer counts were 4.2 ± 3.4 times for sERC+ and 4.1 ± 3.1 times for sERC- blastocyst transfers (p = 0.840). The proportion of high-quality blastocysts was 48.4% for sERC+ transfers and 54.7% in sERC- transfers (p = 0.384). The implantation rates were 24.2% for sERC+ and 32.6% for sERC- transfers (p = 0.198), miscarriage rates were 43.5% vs. 35.5% (p = 0.552), and live birth rates were 13.7% vs. 21.1% (p = 0.172), respectively, with no statistically significant differences. Thirteen live births from sERC+ blastocyst transfers were recorded, and no congenital abnormalities were observed. Limitations, reasons for caution This study exclusively centered on oocytes subjected to ICSI, and the inclusion criteria were restricted to embryos derived from sERC+ oocytes in accordance with clinic policy limitations. Preimplantation genetic testing was not performed on the majority of the transferred embryos. Wider implications of the findings No congenital abnormalities were observed in live births resulting from sERC+ derived blastocyst transfers. However, the limited number of embryo transfers involving sERC+ oocytes poses a constraint in drawing a definitive conclusion regarding the safety of embryos from sERC+ oocytes, and further data is required. Trial registration number non-clinical trials

Response of multipolar retinal neurons to photodamage in the experiment

Purpose: to assess the reaction of multipolar retinal neurons to light irradiation depending on the intensity and duration of exposure. Material and methods. Outbred sexually mature white rats (n = 50, 100 eyes) weighing 180–200 g were exposed to continuous round-the-clock light (200, 3,500 lux; 1, 2, 7, 14, 30 days). The control group consisted of 25 non-irradiated animals (50 eyes). Using semifine sections, colored with toluidine blue, we counted the number of neurons in the ganglionic layer with karyopyknosis, focal and total chromatolysis. In the optic nerve, the percentage of degeneratively altered axons and the number of nerve fibers with deformation of the myelin sheath were calculated. Ultrastructural changes in neurons were studied using a JEM-100 CX-II electron microscope. Results. In the first days of the experiment (1, 2 days), reactive and destructive changes in organelles are observed in the perikaryons of multipolar retinal neurons. The granular endoplasmic reticulum becomes fragmented, loses part of its ribosomes, and vacuoles of varied sizes are formed from its cisterns. With an increase in the duration of exposure (7–30 days), degradation processes are increasing, all the more so after high-intensity (3,500 lux) light irradiation. The photodamage causes changes in all components of the optic nerve and is characterized by a destruction of organelles, a decrease in the number of elements of the cytoskeleton in the axon, and myelin sheath splitting. Conclusion. Changes in multipolar neurons of the retina after photodamage are primarily related to the content and distribution of the chromatophilic substance and depend on the intensity and duration of illumination.

The Relationship Between Smooth Endoplasmic Reticulum Clusters in Metaphase II Oocytes and Embryological and Birth Outcomes in Infertile Couples.

To assess the relationship between oocytes with smooth endoplasmic reticulum cluster (SERc) and embryological and birth outcomes in infertile couples. This was a descriptive study that included 231 infertile patients undergoing in vitro fertilization (IVF) with a total of 2447 mature oocytes (MII), of which 279 oocytes with SERc(+) from 100 patients, the remaining 2168 oocytes with SERc(-). Oocytes were evaluated for the presence or absence of the SERc simultaneously with intracytoplasmic sperm injection at 200x magnification using inverted microscopy - Observe D1. The mean age of patients was 32.05 ± 5.56 years. One hundred patients had at least one SERc(+) oocyte (with 279 SERc(+) and 956 SERc(-) oocytes). One hundred and thirty-one patients had 1212 SERc(-) oocytes. Fertilization outcomes and the rates of good-quality embryos on day 2 and day 5 did not differ between the SERc(+) and the SERc(-) groups. In the first frozen embryo transfer cycles, the clinical pregnancy rate in the group of patients with SERc(+) was not different with the SERc(-) group (61.1% vs 48.78%, p = 0.074, respectively). The live birth rate in the SERc(+) group was statistically significantly higher than the SERc(-) group (57.7% vs 43.9%, p = 0.045, respectively). The fertilization rate, the quality of embryos on days 2 and 5 from oocytes with SERc(+) are similar to those with SERc(-). The live birth rate in the patients with SERc(+) group is statistically significantly higher than the SERc(-) group. There is no difference in clinical pregnancy rate between patients with and without SERc. Therefore, the exclusion of oocytes with SERc should not be recommended.

Ultrastructure of the Bovine Testis in Cattle (Bos taurus): New View.

The purpose of this study was to analyze the ultrastructure of the testes of sexually immature calves and reproductive bulls of the Polish Holstein-Friesian Black-and-White breed. Utilizing TEM, this study identified three distinct stages of seminiferous tubule development in calves, characterized by varying shapes, distributions, and arrangements of individual cells. In immature animals, early developing spermatocytes, prespermatogonia, and pre-Sertoli cells were observed within the seminiferous tubules. In sexually mature bulls, all cells of the spermatogenic series were observed, situated on a thin, multilayered basal lamina, which forms characteristic undulations. An abundant smooth endoplasmic reticulum was observed in the cytoplasm of spermatogonia in both groups of animals, forming characteristic membranous swirls. In adult bulls, spermatogonia maintain contact with each other through numerous cytoplasmic bridges and cell connections, forming small spaces with visible microvilli between them. The ultrastructural analysis facilitated the identification of morphological changes occurring during the maturation of pre-Sertoli cells, transitioning from a large euchromatic nucleus to a nucleus in which the formation of characteristic vesicles and tubules could be observed. It should also be emphasized that two types of Sertoli cells, namely dark and light electron-dense cells, can be found in cattle. These cells differ from each other, indicating that they may perform different functions. The widespread recognition of the presence of two types of Sertoli cells in cattle will undoubtedly contribute to a better understanding of the processes occurring within the testes and provide a basis for further research in this area.

Synapse-specific structural plasticity that protects and refines local circuits during LTP and LTD.

Synapses form trillions of connections in the brain. Long-term potentiation (LTP) and long-term depression (LTD) are cellular mechanisms vital for learning that modify the strength and structure of synapses. Three-dimensional reconstruction from serial section electron microscopy reveals three distinct pre- to post-synaptic arrangements: strong active zones (AZs) with tightly docked vesicles, weak AZs with loose or non-docked vesicles, and nascent zones (NZs) with a postsynaptic density but no presynaptic vesicles. Importantly, LTP can be temporarily saturated preventing further increases in synaptic strength. At the onset of LTP, vesicles are recruited to NZs, converting them to AZs. During recovery of LTP from saturation (1-4 h), new NZs form, especially on spines where AZs are most enlarged by LTP. Sentinel spines contain smooth endoplasmic reticulum (SER), have the largest synapses and form clusters with smaller spines lacking SER after LTP recovers. We propose a model whereby NZ plasticity provides synapse-specific AZ expansion during LTP and loss of weak AZs that drive synapse shrinkage during LTD. Spine clusters become functionally engaged during LTP or disassembled during LTD. Saturation of LTP or LTD probably acts to protect recently formed memories from ongoing plasticity and may account for the advantage of spaced over massed learning. This article is part of a discussion meeting issue 'Long-term potentiation: 50 years on'.

Key Roles of CACNA1C/Cav1.2 and CALB1/Calbindin in Prefrontal Neurons Altered in Cognitive Disorders.

The risk of mental disorders is consistently associated with variants in CACNA1C (L-type calcium channel Cav1.2) but it is not known why these channels are critical to cognition, and whether they affect the layer III pyramidal cells in the dorsolateral prefrontal cortex that are especially vulnerable in cognitive disorders. To examine the molecular mechanisms expressed in layer III pyramidal cells in primate dorsolateral prefrontal cortices. The design included transcriptomic analyses from human and macaque dorsolateral prefrontal cortex, and connectivity, protein expression, physiology, and cognitive behavior in macaques. The research was performed in academic laboratories at Yale, Harvard, Princeton, and the University of Pittsburgh. As dorsolateral prefrontal cortex only exists in primates, the work evaluated humans and macaques. Outcome measures included transcriptomic signatures of human and macaque pyramidal cells, protein expression and interactions in layer III macaque pyramidal cells using light and electron microscopy, changes in neuronal firing during spatial working memory, and working memory performance following pharmacological treatments. Layer III pyramidal cells in dorsolateral prefrontal cortex coexpress a constellation of calcium-related proteins, delineated by CALB1 (calbindin), and high levels of CACNA1C (Cav1.2), GRIN2B (NMDA receptor GluN2B), and KCNN3 (SK3 potassium channel), concentrated in dendritic spines near the calcium-storing smooth endoplasmic reticulum. L-type calcium channels influenced neuronal firing needed for working memory, where either blockade or increased drive by β1-adrenoceptors, reduced neuronal firing by a mean (SD) 37.3% (5.5%) or 40% (6.3%), respectively, the latter via SK potassium channel opening. An L-type calcium channel blocker or β1-adrenoceptor antagonist protected working memory from stress. The layer III pyramidal cells in the dorsolateral prefrontal cortex especially vulnerable in cognitive disorders differentially express calbindin and a constellation of calcium-related proteins including L-type calcium channels Cav1.2 (CACNA1C), GluN2B-NMDA receptors (GRIN2B), and SK3 potassium channels (KCNN3), which influence memory-related neuronal firing. The finding that either inadequate or excessive L-type calcium channel activation reduced neuronal firing explains why either loss- or gain-of-function variants in CACNA1C were associated with increased risk of cognitive disorders. The selective expression of calbindin in these pyramidal cells highlights the importance of regulatory mechanisms in neurons with high calcium signaling, consistent with Alzheimer tau pathology emerging when calbindin is lost with age and/or inflammation.

Volume microscopic analysis of membrane contact sites in mouse kidney renal proximal tubule epithelial cells

Subcellular organelles communicate with one another and with the plasma membrane (PM). A wide variety of membrane contact sites (MCS) tether subcellular structures to one another and mediate inter-organelle communication and exchange. The nature, distribution and function of membrane contact sites within the segment-specific architecture of renal epithelial cells has only just begun to be elucidated. We have used advanced imaging techniques, including Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) and machine learning-based segmentation, to create a comprehensive, high resolution, 3D and quantifiable volume microscopic atlas of the ultrastructure of proximal tubule epithelial cells of the mouse kidney. We applied this data set to identify and quantify MCS between the endoplasmic reticulum and mitochondria and the PM in these cells. FIB-SEM was performed on a 50 μm by 50 μm by 44 μm block of tissue prepared from the renal cortex of a perfusion fixed 12-week-old male mouse. OsO4-stained tissue was embedded in Durcupan and a 3D X-ray tomogram of the block was used to select a volume of interest that included extended lengths of multiple proximal tubule (PT). The volume was imaged by a customized enhanced FIB-SEM system. The X-Y scanning resolution was set to 4-nm, and 4-nm FIB milling was used to expose surfaces for successive imaging. Generation of the complete image stack required ~11,000 cycles of scanning/FIB ablation over ~4 weeks of uninterrupted imaging. Segmentation of the resultant image set was performed with Zeiss Arivis Pro technology. The boundary box for reconstruction had the dimensions of 29.2 μm x 24.8 μm x 8 μm with a volume of 5793.280 μm3 comprising of 2000 consecutive images at an isotropic voxel size of 4 nm. Deep learning segmentation teased apart 10 cells with distinct boundaries in the PT structure. The volume of the cells in our clipped volume ranged from 8.79 μm3 to 1438.53 μm3 with a median of 45.20 μm3. In one representative proximal tubule cell the volume of mitochondria makes up 84.91 μm3 (31.62%) of the cell volume and the volume of Endoplasmic Reticulum (ER) makes up 23.92 μm3 (8.91%) of the cell volume. The 3D reconstruction and quantitative analysis revealed that there is one dominant ER object accounting for most of the ER volume in the whole cell: i.e., the largest ER object takes up 22.37 μm3 or 93.5% of the ER volume in the cell. A post deep-learning processing algorithm used 3D object modifications to identify 11,109 3D MCS between mitochondria and ER in the 10 cells. The Mito-ER MCS ranged from 512 nm3 to 354,304 nm3 with a median volume of 1792 nm3. Visually we can see Mito-ER and Mito-PM MCS extending across a region of up to ~45 nm between the two organelles. Taken together, these data show that the basal lateral surfaces of PT cell PMs are generously invested with fenestrated smooth ER, as are mitochondria and peroxisomes. Extensive regions of direct contact between mitochondria and PM are also observed. The biochemical identities of these contacts and their physiological functions are being actively pursued. NIDDK DK072612 NIDDK DK120534. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Safety and Efficacy of Erbium: Yttrium-Aluminum-Garnet Laser Treatment in Chinese Women with Mild-to-Moderate Stress Urinary Incontinence.

Background: This study aims to evaluate the safety and efficacy of erbium:yttrium-aluminum-garnet (Er:YAG) laser treatment in female patients with mild-to-moderate stress urinary incontinence (SUI). Methods: From July 2018 to June 2020, 72 female patients with mild-to-moderate SUI were enrolled in this study. A baseline assessment was conducted, which included a 1-hour pad test, the validated International Consultation on Incontinence Questionnaire-Urinary Incontinence Short Form (ICIQ-UI-SF), postvoid residual (PVR) testing, pelvic organ prolapse quantification (POP-Q) testing, and a cough stress test. All patients underwent four sessions of Er:YAG laser treatment using a smooth mode. A reassessment was performed 6 months after treatment to evaluate the safety and efficacy of the Er:YAG laser. Results: All patients completed four clinic visits, with a 1-month interval, and were followed up for a minimum of 6 months. No severe adverse reactions were observed during the treatment process. The 1-hour pad test revealed a significant reduction in urinary leakage from baseline (6.30 ± 1.06 g) to the 6-month follow-up (2.70 ± 0.96 g, p < 0.001), with 34 of 72 (47.22%) patients achieving negative results. The ICIQ-UI-SF score significantly decreased from baseline to 6 months (10.82 ± 1.38 to 2.96 ± 0.52, p < 0.001). PVR experimental results showed a significant decrease in residual urine volume after treatment (103.72 ± 8.61 mL to 43.86 ± 4.92 mL, p < 0.001). At the 6-month follow-up, hematoxylin and eosin staining results demonstrated that Er:YAG laser treatment significantly facilitated an increase in the thickness of squamous epithelial cells. The efficacy of Er:YAG laser treatment for SUI was 77.78% (56/72). Conclusions: Several objective and subjective assessments confirmed the safety and efficacy of vaginal smooth mode Er:YAG laser treatment for mild-to-moderate SUI during the 6-month follow-up period. Nonablative Er:YAG laser in the smooth mode is a viable treatment option for SUI patients.

Targeting of Specialized Metabolites Biosynthetic Enzymes to Membranes and Vesicles by Posttranslational Palmitoylation: A Mechanism of Non-Conventional Traffic and Secretion of Fungal Metabolites.

In nature, the formation of specialized (secondary) metabolites is associated with the late stages of fungal development. Enzymes involved in the biosynthesis of secondary metabolites in fungi are located in distinct subcellular compartments including the cytosol, peroxisomes, endosomes, endoplasmic reticulum, different types of vesicles, the plasma membrane and the cell wall space. The enzymes traffic between these subcellular compartments and the secretion through the plasma membrane are still unclear in the biosynthetic processes of most of these metabolites. Recent reports indicate that some of these enzymes initially located in the cytosol are later modified by posttranslational acylation and these modifications may target them to membrane vesicle systems. Many posttranslational modifications play key roles in the enzymatic function of different proteins in the cell. These modifications are very important in the modulation of regulatory proteins, in targeting of proteins, intracellular traffic and metabolites secretion. Particularly interesting are the protein modifications by palmitoylation, prenylation and miristoylation. Palmitoylation is a thiol group-acylation (S-acylation) of proteins by palmitic acid (C16) that is attached to the SH group of a conserved cysteine in proteins. Palmitoylation serves to target acylated proteins to the cytosolic surface of cell membranes, e.g., to the smooth endoplasmic reticulum, whereas the so-called toxisomes are formed in trichothecene biosynthesis. Palmitoylation of the initial enzymes involved in the biosynthesis of melanin serves to target them to endosomes and later to the conidia, whereas other non-palmitoylated laccases are secreted directly by the conventional secretory pathway to the cell wall space where they perform the last step(s) of melanin biosynthesis. Six other enzymes involved in the biosynthesis of endocrosin, gliotoxin and fumitremorgin believed to be cytosolic are also targeted to vesicles, although it is unclear if they are palmitoylated. Bioinformatic analysis suggests that palmitoylation may be frequent in the modification and targeting of polyketide synthetases and non-ribosomal peptide synthetases. The endosomes may integrate other small vesicles with different cargo proteins, forming multivesicular bodies that finally fuse with the plasma membrane during secretion. Another important effect of palmitoylation is that it regulates calcium metabolism by posttranslational modification of the phosphatase calcineurin. Mutants defective in the Akr1 palmitoyl transferase in several fungi are affected in calcium transport and homeostasis, thus impacting on the biosynthesis of calcium-regulated specialized metabolites. The palmitoylation of secondary metabolites biosynthetic enzymes and their temporal distribution respond to the conidiation signaling mechanism. In summary, this posttranslational modification drives the spatial traffic of the biosynthetic enzymes between the subcellular organelles and the plasma membrane. This article reviews the molecular mechanism of palmitoylation and the known fungal palmitoyl transferases. This novel information opens new ways to improve the biosynthesis of the bioactive metabolites and to increase its secretion in fungi.

Content and ultrastructure of natural killer cells in the blood of patients with newly diagnosed type 2 diabetes mellitus

Background. In patients with newly diagnosed type 2 diabetes mellitus (T2DM), a small but statistically significant decrease in absolute number of natural killer (NK) cells (CD56+) in the peripheral blood (PB) was found, especially pronounced with concomitant obesity. In connection with the above, one of the main aims of the present report was to further study the role of NK cells in patients with newly diagnosed T2DM, including those with obesity. For this purpose, a combination of flow cytometry methods with scanning and transmission electron microscopy was used to determine the number and function of CD56+ cells in the PB of patients with T2DM. Materials and methods. We examined 144 patients of both sexes with newly diagnosed T2DM aged 40–70 years who had no other acute or chronic diseases and had not previously taken any hypoglycemic drugs. The control group consisted of 50 healthy individuals with normoglycemia aged 40–65 years who had not COVID-19 disease. Results. Our studies have found that in untreated patients with newly diagnosed T2DM compared to healthy controls, there is a very small statistically significant decrease in the absolute number of CD56+ cells in the PB. When combining flow cytometry (FACS method), transmission and scanning electron microscopy in patients with T2DM, a significant change in the ultrastructure of CD56+ cells was revealed, indicating a decrease in their function (an increase in the number of cells with a smoother surface of the cell membrane, a sharp decrease in the number of azurophilic granules in the cytoplasm considered the main producer of cytokines and perforins, the appearance of parallel tubular structures, biomarkers of decreased cell function, a disruption of the Golgi apparatus structure, granular endoplasmic reticulum, etc.). The data obtained indicate that at the initial stage of T2DM development in humans, there is a pronounced weakening in the function of NK cells and, consequently, the natural immune defense of the body and explain the increased susceptibility of diabetics to various viruses and infections, including COVID-19, and also more severe clinical course and increased mortality. Conclusions. The conducted studies show that in patients with T2DM who are at the initial stage of T2DM, there is a sharp disturbance in the submicroscopic organization of CD56+ cells, the most important cellular element of natural immunity, which suggests a weakening of the function of natural immune defense. It is the main cause of increased sensitivity of diabetic patients to infection with various pathogens, including the SARS-CoV-2, as well as more severe clinical course and increased mortality rate.

Do electromagnetic fields significantly affect thyroid cells and their functions? – A systematic review

Background: This review aimed to emphasize the effects, mechanisms involved, conditions required to trigger impacts, and the adapted responses of EMF on thyroid gland. Methods: The included literatures were collected from the Oceania Radiofrequency Scientific Advisory Association (ORSAA) database which were published before April 2021. The searched was then refined with keyword ‘thyroid’. Results: A total of 15 experimental studies, 10 observational studies, 4 review articles, and 1 medical hypothesis were included. Hypothyroidism is the most reported functional abnormality. The identified morphological abnormalities of the thyroid gland include increased follicular epithelial and interfollicular tissue, decreased colloid volume of cubic cells, follicular fluid, interfollicular space, changes in lysosomes, granular endoplasmic reticulum, cell nuclei, changes in glandular structures including cell hypotrophy, glandular hypertrophy, and increased apoptosis via caspase-dependent pathways. The observational studies reported an increase in thyroid cancer incidence and the diameter of the left anteroposterior of the thyroid gland. Postulation that thyroid cancer is associated with thyroid cancer EMF exposure, is suggested by a medical hypothesis paper. The possibilities of hypothyroidism, thyroid cancer, and cell apoptosis due to EMF exposure have been highlighted in the review articles. Conclusion: Despite these findings, critical elements, such as the effects and mechanisms of EMF exposure on thyroid hormone transporters, genomic and non-genomic actions, conditions required for the effect to occur, and the resultant adapted responses, have not been explored suggesting the need of further research.

Review of ER stress and unfolded protein response

The cell is the fundamental unit of life, and all of the organelles within it perform essential tasks. Cells must meet their typical requirements for survival and development in order to contribute to the functioning of living things. Protein folding in the rough ER, the major organelle for the synthesis, folding, and modification of secreted and transmembrane proteins, is essential for their proper function in the cytoplasm. Endoplasmic reticulum stress (ERS) occurs when the protein-folding capacity of this organelle is disrupted due to various external causes or intracellular events, resulting to a buildup of misfolded or unfolded proteins. Concerns in this area are possible. Some illnesses, cell death, and metabolic abnormalities are examples. The cell's stress response mechanism is the unfolded protein response (UPR). Reduced translation, degradation of unfolded proteins to reduce damage, and increased production of cell-mate proteins all aid in the folding of nascent proteins. This research uses a literature review approach to learn about the ER, an experiment on ER Stress, the reaction mechanism of unfolded protein response (UPR), diseases induced by ER Stress, and the significance of UPR in these illnesses.