Search

Abstract: In the Madeira cockroach, pigment‐dispersing factor‐immunoreactive (PDF‐ir) neurons innervating the circadian clock, the accessory medulla (AME) in the brain′s optic lobes, control circadian behaviour. Circadian activity rhythms are entrained to daily light–dark cycles only by compound eye photoreceptors terminating in the lamina and medulla. Still, it is unknown which neurons connect the photoreceptors to the clock to allow for light entrainment. Here, we characterized by multiple‐label immunocytochemistry the serotonin (5‐HT)‐ir anterior fibre fan and GABA‐ir pathways connecting the AME‐ and optic lobe neuropils. Colocalization of 5‐HT with PDF was confirmed in PDF‐ir lamina neurons (PDFLAs). Double‐labelled fibres were traced to the AME originating from colabelled PDFLAs branching in accessory laminae and proximal lamina. The newly discovered GABA‐ir medial layer fibre tract connected the AME to the medulla′s medial layer fibre system, and the distal tract fibres connected the AME to the medulla. With Ca2+ imaging on primary cell cultures of the AME and with loose‐patch‐clamp recordings in vivo, we showed that both neurotransmitters either excite or inhibit AME clock neurons. Because we found no colocalization of GABA and 5‐HT in any optic lobe neuron, GABA‐ and 5‐HT neurons form separate clock input circuits. Among others, both pathways converged also on AME neurons that coexpressed mostly inhibitory GABA‐ and excitatory 5‐HT receptors. Our physiological and immunocytochemical studies demonstrate that GABA‐ and 5‐HT‐immunoreactive neurons constitute parallel excitatory or inhibitory pathways connecting the circadian clock either to the lamina or medulla where photic information from the compound eye is processed. [ABSTRACT FROM AUTHOR]

A family of four from the Guangxi Zhuang Autonomous Region of China, including a child with α-thalassaemia and hereditary spherocytosis (HS), underwent laboratory identification, and genetic analysis. After harvesting peripheral blood samples from the child patient and his family members, GAP-polymerase chain reaction (PCR) and reverse dot-blot tests were used to identify thalassaemia genotypes. After amplifying exons and the adjacent introns of solute carrier family 4 member 1 (Diego blood group) (SLC4A1), ankyrin 1, spectrin α erythrocytic 1, spectrin β erythrocytic and erythrocyte membrane protein band 4.2 by PCR, DNA sequencing was utilised to detect gene mutations of HS. The thalassaemia gene of the child patient was -α3.7/αα and identical to the genotype of his mother. DNA testing of HS identified two mutation sites on the SLC4A1 gene: Exon 3 c.113A>C (Asp 38 Ala) and intron 7 c.609+86G>A. The father and older sister of the patient also had the same mutations. Due to the mutual interference with disorders of haemoglobin synthesis and erythrocyte membrane defects of laboratory results, it is difficult to diagnose HS when it coexists with thalassaemia. When clinical manifestations and laboratory results cannot be explained by a single haemolytic anaemia, the possibility of combining with another haemolytic anaemia should be considered. Thus, it is necessary to perform pedigree investigation and genetic analyses for a final diagnosis. [ABSTRACT FROM AUTHOR]

Transplantation studies have pinpointed the circadian clock of the Madeira cockroach to the accessory medulla (AME) of the brain’s optic lobes. The AME is innervated by approximately 240 adjacent neuropeptidergic neurons, including 12 pigment-dispersing factor (PDF)-expressing neurons anterior to the AME (aPDFMEs). Four of the aPDFMEs project contralaterally, controlling locomotor activity rhythms of the night-active cockroach. The present in vitro Ca2+ imaging analysis focuses on contralaterally projecting AME neurons and their responses to PDF, GABA, and acetylcholine (ACh). First, rhodamine-dextran backfills from the contralateral optic stalk identified contralaterally projecting AME neurons, which were then dispersed in primary cell cultures. After characterization of PDF, GABA, and ACh responses, PDF immunocytochemistry identified ipsilaterally and contralaterally projecting PDFMEs. All PDF-sensitive clock neurons, PDF-immunoreactive clock neurons, and the majority of ipsilaterally and contralaterally projecting cells were excited by ACh. GABA inhibited all PDF-expressing clock neurons, and about half of other ipsilaterally projecting and most contralaterally projecting clock neurons. For the first time, we identified PDF autoreceptors in PDF-secreting cockroach circadian pacemakers. The medium-sized aPDFMEs and all other contralaterally projecting PDF-sensitive clock cells were inhibited by PDF. The ipsilaterally remaining small PDF-sensitive clock cells were activated by PDF. Only the largest aPDFME did not express PDF autoreceptors. We hypothesize that opposing PDF signaling generates 2 different ensembles of clock cells with antiphasic activity, regulating and maintaining a constant phase relationship between rest and activity cycles of the night-active cockroach. [ABSTRACT FROM AUTHOR]

Fine particulate matter (PM2.5) is a major component of air pollutions that are closely associated with increased risk of lung cancer. However, the role of PM2.5 in the etiology of lung cancer is largely unknown. In this study, we performed acute (24 hours) and chronic (five passages) exposure models to investigate the carcinogenetic mechanisms of PM2.5 by targeting the induction of epithelial-mesenchymal transition (EMT) and cancer stem cells (CSC) properties in human non-small cell lung cancer cell line A549. We found that both acute and chronic PM2.5 exposure enhanced cell migration and invasion, decreased mRNA expression of epithelial markers and increased mRNA expression of mesenchymal markers. Chronic PM2.5 exposure further induced notable EMT morphology and CSC properties, indicating the developing process of cell malignant behaviors from acute to chronic PM2.5 exposure. CSC properties induced by chronic PM2.5 exposure characterized with increased cell-surface markers (CD44, ABCG2), self-renewal genes (SOX2 and OCT4), side population cells and neoplastic capacity. Furthermore, the levels of three stemness-associated microRNAs, Let-7a, miR-16 and miR-34a, were found to be significantly downregulated by chronic PM2.5 exposure, with microarray data analysis from TCGA database showing their lower expression in human lung adenocarcinoma tissues than that in the adjacent normal lung tissues. These data revealed that the induction of EMT and CSC properties were involved in the lung cancer risk of PM2.5, and implicated CSC properties and related microRNAs as possible biomarkers for carcinogenicity prediction of PM2.5. [ABSTRACT FROM AUTHOR]

Electrical activity in the gamma frequency range is instrumental for temporal encoding on the millisecond scale in attentive vertebrate brains. Surprisingly, also circadian pacemaker neurons in the cockroach Rhyparobia maderae ( Leucophaea maderae) employ fast spontaneous rhythmic activity in the gamma band frequency range (20-70 Hz) together with slow rhythmic activity. The ionic conductances controlling this fast spontaneous activity are still unknown. Here, Ca2+ imaging combined with pharmacology was employed to analyse ion channels underlying spontaneous activity in dispersed circadian pacemakers of the adult accessory medulla, which controls circadian locomotor activity rhythms. Fast spontaneous Ca2+ transients in circadian pacemakers accompany tetrodotoxin (TTX)-blockable spontaneous action potentials. In contrast to vertebrate pacemakers, the spontaneous depolarisations from rest appear to be rarely initiated via TTX-sensitive sustained Na+ channels. Instead, they are predominantly driven by mibefradil-sensitive, low-voltage-activated Ca2+ channels and DK-AH269-sensitive hyperpolarisation-activated, cyclic nucleotide-gated cation channels. Rhythmic depolarisations activate voltage-gated Na+ channels and nifedipine-sensitive high-voltage-activated Ca2+ channels. Together with Ca2+ rises, the depolarisations open repolarising small-conductance but not large-conductance Ca2+-dependent K+ channels. In contrast, we hypothesise that P/Q-type Ca2+ channels coupled to large-conductance Ca2+-dependent K+ channels are involved in input-dependent activity. [ABSTRACT FROM AUTHOR]

Pigment-dispersing factor–immunoreactive neurons anterior to the accessory medulla (aPDFMes) in the optic lobes of insects are circadian pacemaker neurons in cockroaches and fruit flies. The authors examined whether any of the aPDFMes of the cockroach Leucophaea maderae are sensitive to changes in period and photoperiod of light/dark (LD) cycles as a prerequisite to adapt to changes in external rhythms. Cockroaches were raised in LD cycles of 11:11, 13:13, 12:12, 6:18, or 18:6 h, and the brains of the adults were examined with immunocytochemistry employing antisera against PDF and orcokinin. Indeed, in 11:11 LD cycles, only the number of medium-sized aPDFMes specifically decreased, while it increased in 13:13. In addition, 18:6 LD cycles increased the number of large- and medium-sized aPDFMes, as well as the posterior pPDFMes, while 6:18 LD cycles only decreased the number of medium-sized aPDFMes. Furthermore, PDF-immunoreactive fibers in the anterior optic commissure and orcokinin-immunoreactive fibers in both the anterior and posterior optic commissures were affected by different lengths of light cycles. Thus, apparently different groups of the PDFMes, most of all the medium-sized aPDFMes, which colocalize orcokinin, respond to changes in period and photoperiod and could possibly allow for the adjustment to different photoperiods. [ABSTRACT FROM PUBLISHER]

Background: The initial randomized, double-blinded, actively controlled, phase III ANEAS study (NCT03849768) demonstrated that aumolertinib showed superior efficacy relative to gefitinib as first-line therapy in epidermal growth factor receptor (EGFR)-mutated advanced non-small cell lung cancer (NSCLC). Metastatic disease in the central nervous system (CNS) remains a challenge in the management of NSCLC. This study aimed to compare the efficacy of aumolertinib versus gefitinib among patients with baseline CNS metastases in the ANEAS study., Methods: Eligible patients were enrolled and randomly assigned in a 1:1 ratio to orally receive either aumolertinib or gefitinib in a double-blinded fashion. Patients with asymptomatic, stable CNS metastases were included. Follow-up imaging of the same modality as the initial CNS imaging was performed every 6 weeks for 15 months, then every 12 weeks. CNS response was assessed by a neuroradiological blinded, independent central review (neuroradiological-BICR). The primary endpoint for this subgroup analysis was CNS progression-free survival (PFS)., Results: Of the 429 patients enrolled and randomized in the ANEAS study, 106 patients were found to have CNS metastases (CNS Full Analysis Set, cFAS) at baseline by neuroradiological-BICR, and 60 of them had CNS target lesions (CNS Evaluable for Response, cEFR). Treatment with aumolertinib significantly prolonged median CNS PFS compared with gefitinib in both cFAS (29.0 vs. 8.3 months; hazard ratio [HR] = 0.31; 95% confidence interval [CI], 0.17-0.56; P < 0.001) and cEFR (29.0 vs. 8.3 months; HR = 0.26; 95% CI, 0.11-0.57; P < 0.001). The confirmed CNS overall response rate in cEFR was 85.7% and 75.0% in patients treated with aumolertinib and gefitinib, respectively. Competing risk analysis showed that the estimated probability of CNS progression without prior non-CNS progression or death was consistently lower with aumolertinib than with gefitinib in patients with and without CNS metastases at baseline. No new safety findings were observed., Conclusions: These results indicate a potential advantage of aumolertinib over gefitinib in terms of CNS PFS and the risk of CNS progression in patients with EGFR-mutated advanced NSCLC with baseline CNS metastases., Trial Registration: ClinicalTrials.gov number, NCT03849768., (© 2024 The Author(s). Cancer Communications published by John Wiley & Sons Australia, Ltd on behalf of Sun Yat‐sen University Cancer Center.)

Relative motion breaks a camouflaged target from a same-textured background, thus eliciting discrimination of a motion-defined object. Ring (R) neurons are critical components in the Drosophila central complex, which has been implicated in multiple visually guided behaviors. Using two-photon calcium imaging with female flies, we demonstrated that a specific population of R neurons that innervate the superior domain of bulb neuropil, termed superior R neurons, encoded a motion-defined bar with high spatial frequency contents. Upstream superior tuberculo-bulbar (TuBu) neurons transmitted visual signals by releasing acetylcholine within synapses connected with superior R neurons. Blocking TuBu or R neurons impaired tracking performance of the bar, which reveals their importance in motion-defined feature encoding. Additionally, the presentation of a low spatial frequency luminance-defined bar evoked consistent excitation in R neurons of the superior bulb, whereas either excited or inhibited responses were evoked in the inferior bulb. The distinct properties of the responses to the two bar stimuli indicate there is a functional division between the bulb subdomains. Moreover, physiological and behavioral tests with restricted lines suggest that R4d neurons play a vital role in tracking motion-defined bars. We conclude that the central complex receives the motion-defined features via a visual pathway from superior TuBu to R neurons and might encode different visual features via distinct response patterns at the population level, thereby driving visually guided behaviors. SIGNIFICANCE STATEMENT Animals could discriminate a motion-defined object that is indistinguishable with a same-textured background until it moves, but little is known about the underlying neural mechanisms. In this study, we identified that R neurons and their upstream partners, TuBu neurons, innervating the superior bulb of Drosophila central brain are involved in the discrimination of high-frequency motion-defined bars. Our study provides new evidence that R neurons receive multiple visual inputs from distinct upstream neurons, indicating a population coding mechanism for the fly central brain to discriminate diverse visual features. These results build progress in unraveling neural substrates for visually guided behaviors., (Copyright © 2023 the authors.)

One important limitation for achieving therapeutic expression of human factor VIII (FVIII) in hemophilia A gene therapy is inefficient secretion of the FVIII protein. Substitution of five amino acids in the A1 domain of human FVIII with the corresponding porcine FVIII residues generated a secretion-enhanced human FVIII variant termed B-domain-deleted (BDD)-FVIII-X5 that resulted in 8-fold higher FVIII activity levels in the supernatant of an in vitro cell-based assay system than seen with unmodified human BDD-FVIII. Analysis of purified recombinant BDD-FVIII-X5 and BDD-FVIII revealed similar specific activities for both proteins, indicating that the effect of the X5 alteration is confined to increased FVIII secretion. Intravenous delivery in FVIII-deficient mice of liver-targeted adeno-associated virus (AAV) vectors designed to express BDD-FVIII-X5 or BDD-FVIII achieved substantially higher plasma FVIII activity levels for BDD-FVIII-X5, even when highly efficient codon-optimized F8 nucleotide sequences were employed. A comprehensive immunogenicity assessment using in vitro stimulation assays and various in vivo preclinical models of hemophilia A demonstrated that the BDD-FVIII-X5 variant does not exhibit an increased immunogenicity risk compared to BDD-FVIII. In conclusion, BDD-FVIII-X5 is an effective FVIII variant molecule that can be further developed for use in gene- and protein-based therapeutics for patients with hemophilia A., Competing Interests: A patent application has been submitted by W.X., B.D., and W.C. for the FVIII-X5 (application number W02014209942A1). W.X., B.D., and W.C. hold equity in Ivygen. J. L. is a full-time employee of Baxalta Innovations GmbH, now part of Takeda. F.H., M. K., M.d.l.R., W. H., B.M.R., F. S., and H.R. were employees of Baxalta Innovations GmbH, now part of Takeda, at the time of the study., (© 2020.)

GABA is the most abundant neurotransmitter in the circadian pacemaker circuits of mammals and insects. In the Madeira cockroach the accessory medulla (AME) in the brain's optic lobes is the circadian clock that orchestrates rest-activity rhythms in synchrony with light dark cycles. Three prominent GABAergic tracts connect the AME to termination sites of compound eye photoreceptors in the lamina and medulla. Parallel GABAergic light entrainment pathways were suggested to either advance or delay the clock for adjustment to changing photoperiods. In agreement with this hypothesis GABA activated or inhibited AME clock neurons, allowing for the distinction of three different GABA response types. Here, we examined which GABA receptors are responsible for these response types. We found that both ionotropic GABA A receptors and metabotropic GABA B receptors were expressed in AME clock cells. Via different signalling pathways, either one of them could account for all three GABA response types. The muscimol-dependently activated GABA A receptor formed a chloride channel, while the SKF 97541-dependently activated GABA B receptor signalled via G-proteins, apparently targeting potassium channels. Expression of chloride exporters or importers determined whether GABA A receptor activation hyper- or depolarized AME neurons. For GABA B receptor responses second messenger gated channels present in the clock cells appeared to decide about the polarity of the GABA response. In summary, circadian clock neurons co-expressed inhibitory and/or excitatory GABA A and GABA B receptors in various combinations, while cotransporter expression and the set of second messenger gated ion channels present allowed for distinct signalling in different clock neurons., (© 2018 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Extensive cohort studies have explored the hazards of particulate matter with aerodynamic diameter 2.5 μm or smaller (PM 2.5 ) to human respiratory health; however, the molecular mechanisms for PM 2.5 carcinogenesis are poorly understood. Long non-coding RNAs (lncRNAs) are involved in various pathophysiological processes. In the present study, we investigated the effect of PM 2.5 on the epithelial-mesenchymal transition (EMT) in lung bronchial epithelial cells and the underlying mechanisms mediated by an lncRNA. Organic extracts of PM 2.5 from Shanghai were used to treat human bronchial epithelial cell lines (HBE and BEAS-2B). The PM 2.5 organic extracts induced the EMT and cell transformation. High levels of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), mediated by NF-κB, were involved in the EMT process. For both cell lines, there was a similar response. In addition, MALAT1 interacted with miR-204 and reversed the inhibitory effect of its target gene, ZEB1, thereby contributing to the EMT and malignant transformation. In sum, these findings show that NF-κB transcriptionally regulates MALAT1, which, by binding with miR-204 and releasing ZEB1, promotes the EMT. These results offer an understanding of the regulatory network of the PM 2.5 -induced EMT that relates to the health risks associated with PM 2.5 .

In the Madeira cockroach, pigment-dispersing factor-immunoreactive (PDF-ir) neurons innervating the circadian clock, the accessory medulla (AME) in the brain's optic lobes, control circadian behaviour. Circadian activity rhythms are entrained to daily light-dark cycles only by compound eye photoreceptors terminating in the lamina and medulla. Still, it is unknown which neurons connect the photoreceptors to the clock to allow for light entrainment. Here, we characterized by multiple-label immunocytochemistry the serotonin (5-HT)-ir anterior fibre fan and GABA-ir pathways connecting the AME- and optic lobe neuropils. Colocalization of 5-HT with PDF was confirmed in PDF-ir lamina neurons (PDFLAs). Double-labelled fibres were traced to the AME originating from colabelled PDFLAs branching in accessory laminae and proximal lamina. The newly discovered GABA-ir medial layer fibre tract connected the AME to the medulla's medial layer fibre system, and the distal tract fibres connected the AME to the medulla. With Ca 2+ imaging on primary cell cultures of the AME and with loose-patch-clamp recordings in vivo, we showed that both neurotransmitters either excite or inhibit AME clock neurons. Because we found no colocalization of GABA and 5-HT in any optic lobe neuron, GABA- and 5-HT neurons form separate clock input circuits. Among others, both pathways converged also on AME neurons that coexpressed mostly inhibitory GABA- and excitatory 5-HT receptors. Our physiological and immunocytochemical studies demonstrate that GABA- and 5-HT-immunoreactive neurons constitute parallel excitatory or inhibitory pathways connecting the circadian clock either to the lamina or medulla where photic information from the compound eye is processed., (© 2018 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Silver nanoparticles (AgNPs) are widely used in health and consumer products that routinely contact skin. However, the biological effects and possible mechanisms of AgNPs on skin remain unclear. Gap junctional intercellular communication (GJIC) plays a critical role in multicellular organisms to maintain tissue homeostasis. The aim of this study is to examine if non-coated AgNPs affect GJIC in human keratinocytes (HaCaT cells), and to identify the possible molecular mechanisms responsible for the effects. GJIC, connexin (Cx)43 protein and mRNA expression, and the effect of siRNA-mediated knockdown of Cx43 on GJIC were assessed. HaCaT cells exposed to non-coated AgNPs at different doses after a 24 hour exposure. To explore further the underlying mechanism, reactive oxygen species and mitogen-activated protein kinase pathway were evaluated after 2, 6, 12 and 24 hours. Our results revealed that non-coated AgNP exposure at subcytotoxic doses increase GJIC partially via Cx43 upregulation. Reactive oxygen species and extracellular signal-regulated kinase and activation of c-Jun N-terminal kinase were involved in the AgNP-induced upregulation of Cx43. This study provides new insight into the potential mechanism of AgNP biological activity., (Copyright © 2017 John Wiley & Sons, Ltd.)

Recombinant adeno-associated viral (rAAV) vectors have been widely used in human gene therapy. One major impediment to its broad application is the inability to produce high-quality vectors in mass quantity. Here, an efficient and scalable suspension cell culture system for the production of rAAV vectors is described. In this system, the AAV trans factors, Rep78, Rep52, VP1, VP2, and VP3, were stably integrated into a single vaccinia virus carrier by maximizing the use of alternative codons between genes with identical amino acids, and the cis rAAV genome was carried by an E1/E3 gene-deleted adenovirus. Infection of improved, E1 integrated, suspension-cultured cells with these two viral vectors resulted in the robust production of rAAV vectors. The newly enhanced system can consistently produce ∼1 × 10 15 genome containing rAAV vectors per liter of suspension cells. Moreover, the capsid composition of rAAV vectors produced by this system is markedly different from those produced using the traditional system in that the VP1 protein is more abundant than the VP2 protein (19:1 versus 1:1). The unique VP1 superabundant rAAV vectors produced in this new system exhibited improved transduction in vivo after intravitreal injection.

Fine particulate matter (PM 2.5 ) is a major component of air pollutions that are closely associated with increased risk of lung cancer. However, the role of PM 2.5 in the etiology of lung cancer is largely unknown. In this study, we performed acute (24 hours) and chronic (five passages) exposure models to investigate the carcinogenetic mechanisms of PM 2.5 by targeting the induction of epithelial-mesenchymal transition (EMT) and cancer stem cells (CSC) properties in human non-small cell lung cancer cell line A549. We found that both acute and chronic PM 2.5 exposure enhanced cell migration and invasion, decreased mRNA expression of epithelial markers and increased mRNA expression of mesenchymal markers. Chronic PM 2.5 exposure further induced notable EMT morphology and CSC properties, indicating the developing process of cell malignant behaviors from acute to chronic PM 2.5 exposure. CSC properties induced by chronic PM 2.5 exposure characterized with increased cell-surface markers (CD44, ABCG2), self-renewal genes (SOX2 and OCT4), side population cells and neoplastic capacity. Furthermore, the levels of three stemness-associated microRNAs, Let-7a, miR-16 and miR-34a, were found to be significantly downregulated by chronic PM 2.5 exposure, with microarray data analysis from TCGA database showing their lower expression in human lung adenocarcinoma tissues than that in the adjacent normal lung tissues. These data revealed that the induction of EMT and CSC properties were involved in the lung cancer risk of PM 2.5 , and implicated CSC properties and related microRNAs as possible biomarkers for carcinogenicity prediction of PM 2.5 ., (© 2017 Wiley Periodicals, Inc.)

Recombinant adeno-associated viral (rAAV) vectors have recently achieved clinical successes in human gene therapy. However, the commonly observed, heavier particles found in rAAV preparations have traditionally been ignored due to their reported low in vitro transduction efficiency. In this study, the biological properties of regular and high-density rAAV serotype 8 vectors, rAAV RD and rAAV HD , were systemically compared. Results demonstrated that both rAAV RD and rAAV HD exhibited similar DNA packaging profiles, while rAAV HD capsids contained fewer VP1 and VP2 proteins, indicating that the rAAV HD particles contained a higher DNA/protein ratio than that of rAAV RD particles. Dynamic light scattering and transmission electron microscopy data revealed that the diameter of rAAV HD was smaller than that of rAAV RD . In vitro, rAAV HD was two- to fourfold less efficient in transduction compared with rAAV RD . However, the transduction performance of rAAV HD and rAAV RD was similar in vivo. No significant difference in neutralizing antibody formation against rAAV RD and rAAV HD was observed, suggesting that the surface epitopes of rAAV RD and rAAV HD are congruent. In summary, the results of this study demonstrate that rAAV RD and rAAV HD are equally competent for in vivo transduction, despite their difference in vitro. Therefore, the use of rAAV HD vectors in human gene therapy should be further evaluated., Competing Interests: Author Disclosure No competing financial interests exist.