Medical resource usage for COVID-19 evaluated using the National Database of Health Insurance Claims and Specific Health Checkups of Japan.

PURPOSE: The coronavirus disease 2019 (COVID-19) pandemic exhibited several different waves threatening global health care. During this pandemic, medical resources were depleted. However, the kind of medical resources provided to each wave was not clarified. This study aimed to examine the characteristics of medical care provision at COVID-19 peaks in preparation for the next pandemic. METHODS: Using medical insurance claim records in Japan, we examined the presence or absence of COVID-19 infection and the use of medical resources for all patients monthly by age group. RESULTS: The wave around August 2021 with the Delta strain had the strongest impact on the working population in terms of hospital admission and respiratory support. For healthcare providers, this peak had the highest frequency of severely ill patients. In the subsequent wave, although the number of patients with COVID-19 remained high, they were predominantly older adults, with relatively fewer patients receiving intensive care. CONCLUSIONS: In future pandemics, we should refer to the wave around August 2021 as a situation of medical resource shortage resulting from the COVID-19 pandemic.

Exchangeable leaders of collectively migrating glioma abuse N-cadherin trafficking.

Collectively migrating cells consist of leaders and followers with different features. In this issue, Kim et al. (https://doi.org/10.1083/jcb.202401057) characterize the leader and follower cells in collective glioma migration and uncover important roles of YAP1/TAZ-mediated regulation of N-cadherin in the leader cells.

Primary Culture of Dissociated Neurons from the Embryonic Cerebral Cortex.

Primary neuronal culture is a valuable in vitro model for analyzing the molecular mechanisms underlying the development and function of neural circuits. In contrast to neurons in vivo, primary cultured neurons can easily be transfected with genes of interest or treated with chemicals such as agonists and inhibitors of a specific target molecule. Furthermore, time-dependent morphological changes, such as the acquisition of neuronal polarity, axon elongation, and dendrite branch formation, can be analyzed by using primary neuronal cultures. Here, we describe a method for preparing a primary culture of neurons from the developing cerebral cortex, together with a method for gene transfer to primary cultured cortical neurons.

Immunocytochemistry of Primary Cultured Cerebral Cortical Neurons.

Immunocytochemistry combined with confocal or superresolution microscopy allows us to observe molecular localization and intracellular structures. However, it is challenging to analyze individual neurons in brain tissue, where neurons are densely packed. In contrast, we can easily observe structures such as the axonal growth cone and dendritic spines in dissociated individual neurons. Thus, the immunocytochemistry of primary cultured neurons is often used because it reflects the in vivo condition at least in part. Here, we describe a method for indirect fluorescence immunocytochemistry of primary cultured neurons from the embryonic cerebral cortex. This involves multiple steps including fixation, permeabilization, and antibody reaction, and in particular, we introduce an optimized protocol for permeabilization to enable the precise localization of target molecules.

CSF1R inhibitor PLX3397 depletes microglia in Mongolian gerbil Meriones unguiculatus, but not in syrian hamster Mesocricetus auratus.

Microglia are the residential immune cells in the central nervous system. Their roles as innate immune cells and regulators of synaptic remodeling are critical to the development and the maintenance of the brain. Numerous studies have depleted microglia to elucidate their involvement in healthy and pathological conditions. PLX3397, a blocker of colony stimulating factor 1 receptor (CSF1R), is widely used to deplete mouse microglia due to its non-invasiveness and convenience. Recently, other small rodents, including Syrian hamsters (Mesocricetus auratus) and Mongolian gerbils (Meriones unguiculatus), have been recognized as valuable animal models for studying brain functions and diseases. However, whether microglia depletion via PLX3397 is feasible in these species remains unclear. Here, we administered PLX3397 orally via food pellets to hamsters and gerbils. PLX3397 successfully depleted gerbil microglia but had no effect on microglial density in hamsters. Comparative analysis of the CSF1R amino acid sequence in different species hints that amino acid substitutions in the juxtamembrane domain may potentially contribute to the inefficacy of PLX3397 in hamsters.

Comparison of 1-year cumulative live birth and perinatal outcomes following single blastocyst transfer with or without preimplantation genetic testing for aneuploidy: a propensity score-matched study.

PURPOSE: We evaluated whether preimplantation genetic testing for aneuploidy (PGT-A) could increase the cumulative live birth rate (CLBR) in patients with recurrent implantation failure (RIF) and recurrent pregnancy loss (RPL). METHODS: The clinical records of 7,668 patients who underwent oocyte retrieval (OR) with or without PGT-A were reviewed for 365 days and retrospectively analyzed. Using propensity score matching, 579 patients in the PGT-A group were matched one-to-one with 7,089 patients in the non-PGT-A (control) group. Their pregnancy and perinatal outcomes and CLBRs were statistically compared. RESULTS: The live birth rate per single vitrified-warmed blastocyst transfers (SVBTs) significantly improved in the PGT-A group in all age groups (P < 0.0002, all). Obstetric and perinatal outcomes were comparable between both groups regarding both RIF and RPL cases. Cox regression analysis demonstrated that in the RIF cases, the risk ratio per OR was significantly lower in the PGT-A group than in the control group (P = 0.0480), particularly in women aged < 40 years (P = 0.0364). However, the ratio was comparable between the groups in RPL cases. The risk ratio per treatment period was improved in the PGT-A group in both RIF and RPL cases only in women aged 40-42 years (P = 0.0234 and P = 0.0084, respectively). CONCLUSION: Increased CLBR per treatment period was detected only in women aged 40-42 years in both RIF and RPL cases, suggesting that PGT-A is inappropriate to improve CLBR per treatment period in all RIF and RPL cases.

Noradrenergic stimulation of α1 adrenoceptors in the medial prefrontal cortex mediates acute stress-induced facilitation of seizures in mice.

Stress is one of the critical facilitators for seizure induction in patients with epilepsy. However, the neural mechanisms underlying this facilitation remain poorly understood. Here, we investigated whether noradrenaline (NA) transmission enhanced by stress exposure facilitates the induction of medial prefrontal cortex (mPFC)-originated seizures. In mPFC slices, whole-cell current-clamp recordings revealed that bath application of picrotoxin induced sporadic epileptiform activities (EAs), which consisted of depolarization with bursts of action potentials in layer 5 pyramidal cells. Addition of NA dramatically shortened the latency and increased the number of EAs. Simultaneous whole-cell and field potential recordings revealed that the EAs are synchronous in the mPFC local circuit. Terazosin, but not atipamezole or timolol, inhibited EA facilitation, indicating the involvement of α1 adrenoceptors. Intra-mPFC picrotoxin infusion induced seizures in mice in vivo. Addition of NA substantially shortened the seizure latency, while co-infusion of terazosin into the mPFC inhibited the effect of NA. Finally, acute restraint stress shortened the latency of intra-mPFC picrotoxin infusion-induced seizures, whereas prior infusion of terazosin reversed this stress-induced shortening of seizure latency. Our findings suggest that stress facilitates the induction of mPFC-originated seizures via NA stimulation of α1 adrenoceptors.

Ablation of microglia does not alter circadian rhythm of locomotor activity.

Microglia, as macrophages in the brain, are responsible for immune responses and synaptic remodeling. Although the function of microglia is regulated by circadian rhythms, it is still unclear whether microglia are involved in the generation and light entrainment of circadian rhythms of behavior. Here, we report that microglial depletion does not alter behavioral circadian rhythms. We depleted ~ 95% of microglia in the mouse brain by PLX3397, a CSF1R inhibitor, and analyzed the effect on the spontaneous behaviors of mice. We found that neither the free-running period under constant darkness nor light entrainment under jet-lag circumstances were influenced by the ablation of microglia. Our results demonstrate that the circadian rhythms of locomotor activity, an important output of the circadian clock in the brain, are likely a phenomenon not produced by microglia.

Rab21 regulates caveolin-1-mediated endocytic trafficking to promote immature neurite pruning.

Transmembrane proteins are internalized by clathrin- and caveolin-dependent endocytosis. Both pathways converge on early endosomes and are thought to share the small GTPase Rab5 as common regulator. In contrast to this notion, we show here that the clathrin- and caveolin-mediated endocytic pathways are differentially regulated. Rab5 and Rab21 localize to distinct populations of early endosomes in cortical neurons and preferentially regulate clathrin- and caveolin-mediated pathways, respectively, suggesting heterogeneity in the early endosomes, rather than a converging point. Suppression of Rab21, but not Rab5, results in decreased plasma membrane localization and total protein levels of caveolin-1, which perturbs immature neurite pruning of cortical neurons, an in vivo-specific step of neuronal maturation. Taken together, our data indicate that clathrin- and caveolin-mediated endocytic pathways run in parallel in early endosomes, which show different molecular regulation and physiological function.

Highly sensitive in vitro cytokine release assay incorporating high-density preculture.

Immunostimulatory effects of monoclonal antibodies (mAb) through binding to Fcγ receptors (FcγR) on immune cells are a likely cause of cytokine release syndrome. However, it is difficult to detect the potential risk of FcγR-dependent cytokine release associated with mAb in the current standard cytokine release assays (CRA), including the air-drying solid-phase method using human peripheral blood mononuclear cells (PBMC). To increase the sensitivity to detect FcγR-dependent cytokine release due to mAb, a high-density preculture (HDC) method was incorporated into the air-drying solid-phase CRA. Here, PBMC were exposed to panitumumab, trastuzumab, rituximab, or alemtuzumab at 0.1, 0.3, 1, and 3 µg/well for 24 or 48 hr under both non-HDC and HDC conditions. T-cell agonists (anti-CD3 mAb, anti-CD28 super-agonist [SA] mAb) were used as reference mAb. Panitumumab, trastuzumab, rituximab, or alemtuzumab induced cytokine release under both non-HDC and HDC conditions, and cytokine release caused by alemtuzumab was more pronounced under HDC conditions. To investigate FcγR involvement in cytokine release associated with panitumumab, trastuzumab, rituximab, and alemtuzumab, CRA of these four mAb were conducted with anti-FcγRI, -FcγRII, or -FcγRIII F(ab')2 fragments. The results showed cytokine release caused by trastuzumab, rituximab, and alemtuzumab was significantly suppressed by anti-FcγRIII F(ab')2 pretreatment, and slightly reduced by anti-FcγRI or anti-FcγRII pretreatment, indicating these mAb induced FcγR (especially FcγRIII)-dependent cytokine release from PBMC. Cytokine release caused by panitumumab was slightly suppressed by anti-FcγRIII F(ab')2 pretreatment. Anti-CD3 mAb and anti-CD28 SA mAb also induced significant release of cytokines under HDC conditions compared with that under non-HDC conditions. In conclusion, CRA incorporating HDC into the air-drying solid-phase method using human PBMC could sensitively capture the FcγR-dependent cytokine release potential of mAb.

Nicotine Enhances Object Recognition Memory via Stimulating α4ß2 and α7 Nicotinic Acetylcholine Receptors in the Medial Prefrontal Cortex of Mice.

Nicotine has been known to enhance recognition memory in various species. However, the brain region where nicotine acts and exerts its effect remains unclear. Since the medial prefrontal cortex (mPFC) is associated with memory, we examined the role of the mPFC in nicotine-induced enhancement of recognition memory using the novel object recognition test in male C57BL/6J mice. Systemic nicotine administration 10 min before training session significantly enhanced object recognition memory in test session that was performed 24 h after the training. Intra-mPFC infusion of mecamylamine, a non-selective nicotinic acetylcholine receptor (nAChR) antagonist, 5 min before nicotine administration blocked the effect of nicotine. Additionally, intra-mPFC infusion of dihydro-ß-erythroidine, a selective α4ß2 nAChR antagonist, or methyllycaconitine, a selective α7 nAChR antagonist, significantly suppressed the nicotine-induced object recognition memory enhancement. Finally, intra-mPFC infusion of nicotine 1 min before the training session augmented object recognition memory in a dose-dependent manner. These findings suggest that mPFC α4ß2 and α7 nAChRs mediate the nicotine-induced object recognition memory enhancement.

Effects of the synthetic cannabinoid 5F-AMB on anxiety and recognition memory in mice.

RATIONALE: N-[[1-(5-fluoropentyl)-1H-indazol-3-yl]carbonyl]-L-valine methyl ester (5F-AMB) is a synthetic cannabinoid that has been distributed recently. Although inhalation of 5F-AMB produces adverse effects, such as impaired memory and disturbed consciousness, in humans, the psychopharmacological effects of 5F-AMB in rodents have not been investigated. OBJECTIVES: We first examined the effects of intraperitoneal and intracerebroventricular injections of 5F-AMB on anxiety-like behavior and locomotor activity in the open field (OF) test and recognition memory in the novel object recognition test (NOR) in C57BL/6J mice. We also examined whether a cannabinoid 1 (CB1) receptor antagonist AM251 blocks the effects of 5F-AMB. We next examined the effects of 5F-AMB infusion into the medial prefrontal cortex (mPFC), a brain region associated with anxiety and memory, on these tests. RESULTS: Intraperitoneal injection of 5F-AMB (0.3 mg/kg) dramatically decreased locomotor activity in the OF, and this effect was partially reversed by AM251 (3 mg/kg). Intracerebroventricular infusion of 5F-AMB (10 nmol) produced an anxiolytic effect in the OF and impaired acquisition, but not retrieval, of recognition memory in the NOR, and these effects were blocked by co-infusion of AM251 (1.8 nmol). Bilateral intra-mPFC infusion of 5F-AMB (10 pmol/side) similarly produced impaired recognition memory acquisition, but no anxiolytic effect. CONCLUSIONS: The results demonstrate that centrally administered 5F-AMB produces anxiolytic effect and impaired recognition memory acquisition via activation of CB1 receptors, while systemic 5F-AMB severely impaired locomotor activity. The mPFC is involved in 5F-AMB-induced impairment of recognition memory acquisition. However, other brain region(s) may contribute to the 5F-AMB-induced anxiolytic effect.

Cell migration is negatively modulated by ABCA1.

Temporal and spatial changes of membrane lipid distribution in the plasma membrane are thought to be important for various cellular functions. ATP-Binding Cassette A1 (ABCA1) is a key lipid transporter for the generation of high density lipoprotein. Recently, we reported that ABCA1 maintains an asymmetric distribution of cholesterol in the plasma membrane. Here we report that ABCA1 suppresses cell migration by modulating signal pathways. ABCA1 knockdown in mouse embryonic fibroblasts accelerated cell migration and increased activation of Rac1 and its localization to detergent-resistant membranes. Phosphorylation of MEK and ERK also increased. Inhibition of Rac1 or MEK-ERK signals suppressed cell migration in ABCA1 knockdown cells. Because our experimental conditions for cell migration did not contain cholesterol or lipid acceptors for ABCA1, cellular cholesterol content was not changed. These data suggest that ABCA1 modulates cell migration via Rac1 and MEK-ERK signaling by altering lipid distribution in the plasma membrane.

The synthetic cannabinoid 5F-AMB changes the balance between excitation and inhibition of layer V pyramidal neurons in the mouse medial prefrontal cortex.

RATIONALE: 5F-AMB is one of the synthetic cannabinoids (SCs) designed to potentiate the ability to activate cannabinoid 1 (CB1) receptors and is abused worldwide. Although inhalation of 5F-AMB elicits serious adverse effects including impaired memory and consciousness, it is not known whether and how 5F-AMB affects the activity of pyramidal neurons in the medial prefrontal cortex (mPFC), a brain region associated with higher functions such as memory and cognition. OBJECTIVES: In the present study, we examined the effects of 5F-AMB on mPFC layer V (L5) pyramidal neurons using in vitro whole-cell patch-clamp recordings. RESULTS: Bath application of 5F-AMB attenuated the frequency but not the amplitude of spontaneous excitatory and inhibitory postsynaptic currents (sEPSCs and sIPSCs). The attenuating effects of 5F-AMB were abolished by the CB1 receptor antagonist AM251. 5F-AMB also attenuated the frequency of miniature EPSCs and IPSCs recorded in the presence of tetrodotoxin. Moreover, the extent of attenuating effects of 5F-AMB on stimulus-evoked EPSCs was significantly larger than that on evoked IPSCs. CONCLUSIONS: These findings suggest that 5F-AMB attenuates both excitatory and inhibitory transmission in mPFC L5 pyramidal neurons via the activation of CB1 receptors located in presynaptic terminals. Further, the net impact of 5F-AMB on L5 pyramidal neurons is inhibition due to the change in balance between excitation and inhibition. This inhibitory effect might at least partly contribute to the expression of the adverse effects induced by 5F-AMB inhalation.

ABCA1, ABCG1, and ABCG4 are distributed to distinct membrane meso-domains and disturb detergent-resistant domains on the plasma membrane.

ATP-binding cassette A1 (ABCA1), ABCG1, and ABCG4 are lipid transporters that mediate the efflux of cholesterol from cells. To analyze the characteristics of these lipid transporters, we examined and compared their distributions and lipid efflux activity on the plasma membrane. The efflux of cholesterol mediated by ABCA1 and ABCG1, but not ABCG4, was affected by a reduction of cellular sphingomyelin levels. Detergent solubility and gradient density ultracentrifugation assays indicated that ABCA1, ABCG1, and ABCG4 were distributed to domains that were solubilized by Triton X-100 and Brij 96, resistant to Triton X-100 and Brij 96, and solubilized by Triton X-100 but resistant to Brij 96, respectively. Furthermore, ABCG1, but not ABCG4, was colocalized with flotillin-1 on the plasma membrane. The amounts of cholesterol extracted by methyl-ß-cyclodextrin were increased by ABCA1, ABCG1, or ABCG4, suggesting that cholesterol in non-raft domains was increased. Furthermore, ABCG1 and ABCG4 disturbed the localization of caveolin-1 to the detergent-resistant domains and the binding of cholera toxin subunit B to the plasma membrane. These results suggest that ABCA1, ABCG1, and ABCG4 are localized to distinct membrane meso-domains and disturb the meso-domain structures by reorganizing lipids on the plasma membrane; collectively, these observations may explain the different substrate profiles and lipid efflux roles of these transporters.

[Relationship between red blood cell transfusion volume and posttransfusional iron overload in hematological diseases].

We retrospectively investigated the status of transfusional iron overload at Kinki University Hospital. One hundred and sixty three patients received more than 10 red blood cell (RBC) units per year in 2009 and 2010. Myelodysplastic syndrome (37.4%) and aplastic anemia (11.0%) accounted for about 50% of the underlying diseases. At the time of receiving a total of 20 RBC units, 90.8% and 66.2% of the 65 patients evaluated had more than 500 and 1,000 ng/ml of serum ferritin, respectively. The frequency of organ dysfunction associated with iron overload was 56.9% of all the patients assessed, 37.8% of patients with serum ferritin levels of 500∼999 ng/ml, and 67.4% of patients with serum ferritin levels >1,000 ng/ml. Although the Japanese guidelines propose 40 units of RBC transfusion and/or a serum ferritin level of 1,000 ng/ml as a good point to start iron chelation therapy, our results suggest that iron overload and consequent organ dysfunction may occur earlier than this. Therefore, it may be necessary to start iron chelation therapy earlier than that suggested by the Japanese guidelines.

Importance of uncharged polar residues and proline in the proximal two-thirds (Pro107-Ser128) of the highly conserved region of mouse ileal Na+-dependent bile acid transporter, Slc10a2, in transport activity and cellular expression.

BACKGROUND: SLC10A2-mediated reabsorption of bile acids at the distal end of the ileum is the first step in enterohepatic circulation. Because bile acids act not only as detergents but also as signaling molecules in lipid metabolism and energy production, SLC10A2 is important as the key transporter for understanding the in vivo kinetics of bile acids. SLC10A family members and the homologous genes of various species share a highly conserved region corresponding to Gly104-Pro142 of SLC10A2. The functional importance of this region has not been fully elucidated. RESULTS: To elucidate the functional importance of this region, we previously performed mutational analysis of the uncharged polar residues and proline in the distal one-third (Thr130-Pro142) of the highly conserved region in mouse Slc10a2. In this study, proline and uncharged polar residues in the remaining two-thirds of this region in mouse Slc10a2 were subjected to mutational analysis, and taurocholic acid uptake and cell surface localization were examined. Cell surface localization of Slc10a2 is necessary for bile acid absorption. Mutants in which Asp or Leu were substituted for Pro107 (P107N or P107L) were abundantly expressed, but their cell surface localization was impaired. The S126A mutant was completely impaired in cellular expression. The T110A and S128A mutants exhibited remarkably enhanced membrane expression. The S112A mutant was properly expressed at the cell surface but transport activity was completely lost. Replacement of Tyr117 with various amino acids resulted in reduced transport activity. The degree of reduction roughly depended on the van der Waals volume of the side chains. CONCLUSIONS: The functional importance of proline and uncharged polar residues in the highly conserved region of mouse Slc10a2 was determined. This information will contribute to the design of bile acid-conjugated prodrugs for efficient drug delivery or SLC10A2 inhibitors for hypercholesterolemia treatment.

Evaluation of primary and secondary responses to a T-cell-dependent antigen, keyhole limpet hemocyanin, in rats.

To develop a rat T-cell-dependent antibody response (TDAR) model evaluating both primary and secondary antibody responses, keyhole limpet hemocyanin (KLH) was used to immunize rats twice during a 14-day course of study, a pattern closely linked to that of a short-term general toxicity study. Female rats of four representative strains (e.g., Sprague-Dawley, Wistar, Fischer, and Lewis) were immunized twice with intravenous administrations of KLH (300 µg/rat) on Days 5 and 9 during a 14-day treatment regimen with cyclophosphamide (CPA) at 1, 3, or 6 mg/kg/day. The primary and secondary immunizations of KLH markedly elevated serum anti-KLH IgM and IgG levels in all strains on Days 9 and 15. Remarkable higher levels of anti-KLH IgG (≈ 1000 µg/ml) were noted in all strains, which were more than 4-times compared with those of anti-KLH IgM levels at Day 9, indicating that predominant IgG reactions were induced by the dual immunizations. A large inter-individual variability in KLH-specific IgM and IgG production was observed in all strains. However, levels of the KLH-specific antibodies were considered sufficient for the evaluation, even in Sprague-Dawley and Wistar rats reported as strains with a wide range of variability since immunosuppression of CPA on responses in both anti-KLH IgM and IgG were observed in all strains to the same extent. In addition, the sensitivity of the KLH-ELISA assay system detecting the immunosuppressive effects of CPA was comparable to other assay systems with PFC assay or ELISA using SRBC. The results here demonstrated that these experimental designs could provide valuable information about the influence on both the primary and secondary humoral immune responses in rats when exposed to potential immunomodulatory drugs. Furthermore, the design of the presented TDAR study would support comprehensive evaluation together with the outcome of the conventional general toxicity study.

Supplementation of DHA-rich microalgal oil or fish oil during the suckling period in mildly n-3 fatty acid-deficient rat pups.

Long-chain polyunsaturated fatty acids (LC-PUFA), particularly arachidonic acid (ARA) and docosahexaenoic acid (DHA), are considered critical for the development of infants and are commonly supplemented in infant formulae. In this study, two common sources of n-3 LC-PUFA, fish oil (FO) and DHA-rich microalgal oil (DMO), were fed to rat pups of mildly n-3 PUFA-deficient dams to compare changes in LC-PUFA of tissue phospholipids. The milk from dams fed a n-3 PUFA-deficient diet contained less n-3 LC-PUFA than that of dams fed a control diet (AIN-93G). The pups' were given orally 1 mg/g weight of either FO or DMO for 17 days between the ages of 5 and 21 days, the pups were weaned, and sacrificed 1 week later for analysis of fatty acid compositions of brain, heart, kidney, spleen, and thymus phospholipids. Although both FO and DMO brought about a recovery in the tissue DHA levels compared to those of the control group (pups from AIN-93G-fed dams), DMO was more effective at restoring tissue LC-PUFA status because it was richer in DHA than FO. FO had a slightly lower PUFA level than that required to bring the LC-PUFA status completely to normal levels in this experiment, and EPA did not accumulate in tissues under the conditions tested here. These results demonstrate the effectiveness of ingesting either FO or DMO in the pre-weaning period for improving mild n-3 PUFA deficiency.